Regulators of type-1 tumor necrosis factor receptor and other cytokine receptor shedding

ABSTRACT

The present invention provides compositions and methods for the regulation of cytokine signaling through the Tumor Necrosis Factor (TNF) pathway. Specifically, the invention provides a novel gene, polypeptide and related compositions and methods for the regulation of ectodomain shedding. In preferred embodiments, methods and compositions for the regulation of TNF Type-1 Receptor ectodomain shedding are provided. The present invention finds use in therapeutics, diagnostics, and drug screening applications.

This application is the national stage entry of PCT/US01/06464, filedFeb. 28, 2001, which claims priority benefit to U.S. Provisional PatentApplication No. 60/185,586, filed Feb. 28, 2000.

This invention was made during the course of work supported by theUnited States Government under the National Institutes of Health. Assuch, the United States Government may have certain rights to thisinvention.

FIELD OF THE INVENTION

The present invention provides compositions and methods related toregulation of cytokine signaling through the Tumor Necrosis Factor (TNF)pathway. Specifically, the invention provides novel genes, polypeptidesand related compositions and methods for the regulation of TNF Type-1Receptor ectodomain shedding. It is contemplated that the compositionsand methods of the present invention will also find use in theregulation of ectodomain shedding of other cytokine receptors. It isfurther contemplated that these compositions and methods will find usein therapeutics for the treatment of diseases and disorders of theimmune system.

BACKGROUND OF THE INVENTION

Aberrant regulation of cytokine signaling results in a wide variety ofhyper-inflammatory, autoimmune and immune-deficiency pathologicalconditions. Cytokines are a large and diverse group of molecules whichmediate interactions between cells, ultimately regulating the widevariety of cells of the immune repertoire. Cytokine signaling mediatesnumerous facets of normal immune system physiology, includingdevelopment, response, activation, maintenance, memory and apoptosis(Roitt et al. (Eds.), Immunology, Fifth Edition, Mosby InternationalPublishers [1998]).

Tumor Necrosis Factor

Tumor necrosis factor-α (commonly written as TNF, but also written astumor necrosis factor or TNFα) is a multifunctional cytokine mediatingpleiotropic biological functions in both health and disease states. TNFis secreted primarily by monocytes and macrophages, but can also besecreted by other cell types. The list of processes regulated by TNF isextensive, and includes inflammation, immunoregulation, cyytotoxicityand antiviral effects (See e.g., Vilcek et al, J. Biol. Chem.,266:7313–7316 [1991]). TNF plays an integral role in destroying tumors,mediating responses to tissue injury, and protecting hosts frominfections by various microorganisms (Vassali, Ann. Rev. Immunol.,10:411–452 [1992]). TNF also induces the transcriptional activation ofnumerous genes, including NF-κB and AP-1, with the consequent expressionof pro-inflammatory and immunoregulatory genes (Rothe et al., Cell83:1243–124 [1995]; Varfolomeev et al., J. Exp. Med., 183:1271–1275[1996]; Chinnaiyan et al., J. Biol. Chem., 271:4961–4965 [1996]; Hsu etal., Immunity 4:387–396 [1996]; and Hsu et al., Cell 84:299–308 [1996]).TNF-mediated NF-κB activation is also an important negative feedbackmechanism regulating apoptosis (Beg and Baltimore, Science 274:782–784[1996]; Van Antwerp et al., Science 274:787–789 [1996]; and Wang et al.,Science 274:784–787 [1996]).

TNF in Disease

TNF has also been implicated in the pathogenesis of a variety ofdiseases and disorders. It is theorized that these pathologies resultfrom the aberrant regulation of TNF activity, in which the pathologiesmanifest as a result of excessive or insufficient TNF activity. Amongthe activities for which TNF is most noted are its pro-inflammatoryactions, sometimes termed the “acute phase immune response.”Unfortunately, if not properly regulated, these proinflammatoryresponses can result in tissue injury and chronic inflammatory diseases,such as rheumatoid arthritis, inflammatory bowel disease, septic shock,cachexia, autoirumune disorders, graft-versus-host disease and insulinresistance (Piguet et al., J. Exp. Med., 166:1280 [1987]; Pujol-Borrellet al., Nature 326:304–306 [1987]; Tracey et al., Nature 330:662–664[1987]; Oliff, Cell 54:141–142 [1988]; Vilcek et al., J. Biol. Chem.,266:7313–7316 [1991]; and Eigler et al., Immunol. Today 18:487–92[1997]). Excessive TNF activity results in the detrimental effects of anexaggerated immune response demonstrated in some of these diseases,exemplified by overstimulation of interleukin-6 andgranulocyte/macrophage-colony stimulating factor (GM-CSF) secretion,enhanced cytotoxicity of polymorphonuclear neutrophils, prolongedexpression of cellular adhesion molecules, induction of procoagulantactivity on vascular endothelial cells, increased adherence ofneutrophils and lymphocytes, and stimulation of the release of plateletactivating factor from macrophages, neutrophils and vascular endothelialcells (Vassali, Ann. Rev. Immunol., 10:411–452 [1992]; Vilcek et al., J.Biol. Chem., 266:7313–7316 [1992]; and Barbara et al., Immunol. and CellBiol., 74:434–443 [1996]).

Recent evidence also implicates TNF activity in the pathogenesis of manyinfections. TNF is thought to play a central role in thepathophysiological consequences of Gram-negative sepsis and endotoxicshock, including fever, malaise, anorexia, and cachexia (Beutler et al.,Nature 316:552–554 [1985]; Bauss et al, Infect. Immun., 55:1622–1625[1987]; Tracey et al., Nature 330:662–664 [1987]; and Vassali, Ann. Rev.Immunol., 10:411–452 [1992]). Because TNF can mimic many of thebiological effects of endotoxin, it is theorized that TNF is a centralmediator responsible for the clinical manifestations ofendotoxin-related and other critical illnesses (Waage et al., Lancet1:355–357 [1987]; Cerami et al., Immunol. Today 9:28 [1988]; Mitchie etal., N. Eng. J. Med., 318:1481–1486 [1988]; Revhaug et al., Arch. Surg.,123:162–170 [1988]; and Michie et al., Ann. Surg., 209:19–24 [1989]).

Tumor Necrosis Factor Receptor

The numerous biological effects of TNF are now known to be mediated bytwo transmembrane receptors, the 55 kilodalton Type I receptor (alsowritten as “CD120a,” and referred to herein as “TNFR1”) and the 75kilodalton Type II receptor (also written as “CD120b,” and referred toherein as TNFR2). Although both TNFR1 and TNFR2 demonstrate strongaffinity for TNFα, these two receptors demonstrate no apparent homologyin their cytoplasmic (i.e., intracellular) domains. This fact isconsistent with the observation that these two receptors transducedifferent signals to the nucleus via distinct signaling intermediates(Lewis et al., Proc. Natl. Acad. Sci. USA 88:2830–2834 [1991]; Tartagliaand Goeddel, Immunol. Today 13:151–153 [1992]; and Barbara et al.,Imunol. Cell Biol., 74:434–443 [1996]).

Soluble TNF inhibitors have been identified in normal human urine, aswell as in sera and other body fluids of patients with infectious,neoplastic and immunologic disorders. This observation ultimately led tothe revelation that these soluble TNF inhibitors were actually theextracellular domains of TNF receptors derived by proteolytic cleavageof the transmembrane forms (Engelmann et al., J. Biol. Chem.,264:11974–11980 [1989]; Olsson et al., Eur. J. Haematol., 42:270–275[1989]; Seckinger et al., J Biol. Chem., 264:11966–11973 [1989];Engelmann et al., J. Biol. Chem., 265:1531–1536 [1990]; and Aderka etal., J. Exp. Med., 175:323–329 [1992]). In the case of the TNFR1, thisproteolytic activity results in the cleavage and shedding of theextracellular N-terminal domain (also called the ectodomain). Thesefree, soluble TNFR1 ectodomains (“sTNFR1s”) have an affinity for TNFthat is similar to that of intact membrane receptors. Due to thisaffinity, the free receptors are able to bind and sequester TNF, therebyinhibiting the biological action of TNF. Furthermore, the generation ofsTNFR1 is also likely to suppress TNF signaling by reducing the numberof functional TNF receptors acting at the cell membrane. The sTNFR1ectodomains are also theorized to serve a more complex bufferingfunction in the regulation of TNF activity (Aderka et al., J. Exp. Med.,175:323–329 [1992]; and Werb and Yan, Science 282:1279–1280 [1998]). Thecomplexity of TNF signaling is fuirther illustrated by the observationthat many of the stimuli that result in TNF release also result in therelease of the soluble TNF receptor, suggesting that these soluble TNFinhibitors may serve as part of a regulated feedback mechanism tocontrol TNF activity (Adreke et al., J. Exp. Med., 175:323–329 [1992];and Porteu and Nathan, J. Exp. Med., 172:599–607 [1990]).

The importance of TNFR1 in the regulation of TNF activity in hostdefense, immunoregulation and development has been fuirther demonstratedin studies utilizing TNFR1 knockout mice. Mice deficient in TNFR1 show avariety of phenotypes, including phenotypes which mimic human immunedisorders (Pfeffer et al., Cell 73:457–467 [1993]; Rothe, Nature364:798–802 [1993]; Le Hir et al., J. Exp. Med., 183:2367–2372 [1996];Matsumoto et al., Science 271:1289–1291[1996]; Mori et al. J. Immunol.157:3178–3182 [1996]; Speiser et al., J. Immunol., 158:5185–5190 [1997];Tkachuk et al., J. Exp. Med., 187:469–477 [1998]; and Kagi et al., J.Immunol., 162:4598–4605 [1999]).

The key role of TNFR1 shedding in the regulation of TNF bioactivity ishighlighted by the association of germline mutations in TNFR1extracellular domains with impaired TNFR1 shedding and autoinflammatorydisease characterized by autosomal dominant periodic fever syndromes(McDermott et al., Cell 97:133–144 [1999]).

Other Mediators of Acute Phase Response

In addition to TNF, other cytokines have been implicated in theinduction of the pro-inflammatory response (i.e., the acute phase immuneresponse). These cytokines which demonstrate overlapping activities withTNF include the interleukins (e.g., IL-1 and IL-6) (Suffredini et al.,J. Clin. Immunol., 19:203–214 [1999]).

IL-1 (consisting of both α and β forms) is an important proinflammatorycytokine which regulates the expression of a wide variety of targetgenes and proteins in nearly every cell type (Dinarello, Blood77:1627–1652 [1991]; and Dinarello, The Cytokine Handbook (ed. Angus W.Thomson), 3^(rd) edition, Academic Press, San Diego, p. 35–72 [1998]).The spectrum of IL-1-mediated biologic effects includes inflammatory,metabolic, physiologic, hematopoietic, and immunologic functions. IL-1is thought to play a role in the pathogenesis of several disease states,including septic shock, rheumatoid arthritis, inflammatory boweldisease, myelogenous leukemia, diabetes mellitus, and atherosclerosis(Dinarello et al., N. Engl. J. Med., 328:106–113 [1993]).

IL-6 is also a multifunctional cytokine with pleiotropicpro-inflammatory effects (DiCosmo, et al., J. Clin. Invest.,94:2028–2035 [1994]; and Kishimoto et al., Blood 86:1243–1254 [1995]).For example, IL-6 plays an important role in regulating B cellimmunoglobulin production, T-cell activation, growth anddifferentiation, hematopoiesis, hepatic acute phase reactions andosteoclast development (Hirano, The Cytokine Handbook (ed. Angus W.Thomson), 3^(rd) edition, Academic Press, San Diego, p. 197–228 [1998]).Dysregulated production of IL-6 may contribute to the pathogenesis of avariety of inflammatory, neoplastic and autoimmune disorders, such asplasma cell neoplasia and Castleman's disease (Yoshizaki, et al., Blood74:1360–1367 [1989]; and Hirano, Int. J. Cell Cloning 9:166–184 [1991]).

The signal transduction pathways utilized by TNF, IL-1 and IL-6 alsoshow shared signaling intermediates. For example, both TNF and IL-1 canactivate both NF-κB and AP-1, which are important pro-inflammatorytranscription factors (Ashkenazi et al., Science 281:1305–1308 [1998];and Dinarello, The Cytokine Handbook (ed. Angus W. Thomson), 3^(rd)edition, Academic Press, San Diego, p. 35–72 [1998]). Similarly, IL-6signaling uses components of the JAK-STAT pathway, which has also beenreported to be induced by TNF (Guo et al., J. Immunol., 160:2742–2750[1998]; and Hirano, The Cytokine Handbook (ed. Angus W. Thomson), 3^(rd)edition, Academic Press, San Diego, p. 197–228 [1998]).

The cognate receptors for the IL-1 and IL-6 cytokines are known. Thereare two IL-1 receptor forms, type I and type II. There is a single IL-6receptor, consisting of gp80 alpha chain and gp130 beta chain subunits,where ligand binding is mediated by the alpha subunit. The IL-1 and IL-6receptors are also present as soluble forms analogous to the solubleform of TNFR1. Furthermore, it has been suggested that these receptorsplay a role in the regulation of IL-1 and IL-6 activity andpro-inflammatory response (Dower et al., J. Immunol., 142:4314 [1989];Novick et al., J. Exp. Med., 170:1409 [1989]; Eastgate et al., FEBSLett., 260:213 [1990]; Giri et al., J. Biol. Chem., 265:17416 [1990];Symons et al., Cytokine 2:190 [1990]; Symons et al., FEBS Lett., 272:133[1990]; Symons et al., J. Exp. Med., 174:1251–1254 [1991]; Mullberg etal., Biochem. Biophys. Res. Commun., 189:794 [1992]; Mullberg et al.,Eur. J. Immunol., 23:473 [1993]; Svenson et al., Cytokine 5:427 [1993];and Arend et al., J. Immunol., 153:4766–4774 [1994]).

Analogy between regulation of TNF and other cytokines is furtherillustrated by studies utilizing peptide-hydroxamate metalloproteaseinhibitors. Specifically, the protease inhibitors TAPI (TNF-α proteaseinhibitor) and RU36156 have been reported to inhibit the proteolyticcleavage and shedding of both TNFR1 and IL-6R (Mullberg et al., J.Immunol., 155:5198–5205 [1995]; and Gallea-Robache et al., Cytokine9:340–346 [1997]).

As discussed above, in view of the importance of TNF, IL-1 and IL-6 inboth health and disease states, there exists a need for methods andcompositions for the regulation of TNF, IL-1 and IL-6 cytokine activity.These methods and compositions will find use as therapeutic agents forthe treatment of disease states.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods related toregulation of cytokine signaling through the Tumor Necrosis Factor (TNF)pathway, as well as other signaling pathways controlled by othercytokines, including IL-1 and IL-6. It is contemplated that thesecompositions and methods will find use in therapeutics for the treatmentof diseases and disorders of the immune system.

The present invention also provides novel polypeptides and a nucleicacid sequences. In particular, the present invention provides isolatednucleic acids comprising the nucleotide sequence set forth in SEQ IDNO:1, which encodes a polypeptide referred to as “ARTS-”1 (i.e.,aminopeptidase regulator of type I, 55 kDa tumor necrosis factorreceptor ectodomain shedding) which has the ability to promote theshedding of the extracellular domain of Type I Tumor Necrosis FactorReceptor (TNFR1). Thus, the present invention also provides the isolatedpolypeptides comprising the amino acid sequence set forth in SEQ IDNO:2, as well as isolated nucleic acids encoding the polypeptide setforth in SEQ ID NO:2.

The present invention further provides recombinant vectors comprisingthe ARTS-1 gene and host cells comprising these vectors. In oneembodiment, the host cell is eukaryotic, while in an alternativeembodiment, the host cell is prokaryotic. The present invention alsoprovides antibodies raised against at least a portion of the ARTS-1polypeptide of SEQ ID NO:2. In some embodiments, the antibodies aremonoclonal, while in alternative embodiments, the antibodies arepolyclonal.

In other embodiments, the present invention provides isolated nucleicacids that are substantially homologous to the nucleic acid of SEQ IDNO:1, wherein the nucleic acid is capable of hybridizing under highstringency conditions to the nucleic acid of SEQ ID NO:1. In a preferredembodiment, the nucleic acid substantially homologous to the ARTS-1 geneencodes a polypeptide having the ability to regulate the shedding of theextracellular domain of at least one cytokine receptor. In particularlypreferred embodiments, the cytokine receptor is selected from the groupconsisting of type-1 tumor necrosis factor receptor, type Iinterleukin-1 cytokine receptor, type II interleukin-1 cytokinereceptor, and interleukin-6 cytokine receptor alpha-chain gp80. In someembodiments, the nucleic acid substantially homologous to the nucleicacid of the ARTS-1 gene is identified using PCR methods. In alternativeembodiments, the nucleic acid substantially homologous to the ARTS-1gene is identified using hybridization screening methods.

The present invention further provides methods for the isolation ofamplifiable nucleic acid substantially homologous to the nucleic acid ofSEQ ID NO:1 comprising: providing a sample comprising template nucleicacid suspected of encoding a gene substantially homologous to thenucleic acid of SEQ ID NO:1; and at least two primers; annealing theprimers to the template nucleic acid; extending the primers (e.g., withreiterated DNA synthesis) under conditions such that the templatenucleic acid is amplified, to produce an amplified product; andvisualizing the amplified product. In some preferred embodiments, theamplified product is isolated. The present invention further providesthe product of these amplification methods. In preferred embodiments,the amplified product encodes a polypeptide having the ability toregulate the shedding of the extracellular domain of at least onecytokine receptor. In particularly preferred embodiments, the cytokinereceptor is selected from the group consisting of type-1 tumor necrosisfactor receptor, type I interleukin-1 cytokine receptor, type IIinterleukin-1 cytokine receptor, and interleukin-6 cytokine receptoralpha-chain gp80.

The present invention also provides methods for the use of thesecompositions to regulate the shedding of sTNFR1. It is contemplated thatmethods which regulate the shedding of the sTNFR1 also regulate theactivity of TNF. In a most preferred embodiment, the invention providesmethods for use of these compositions in therapeutic applications in thetreatment of immune system diseases and disorders resulting fromaberrant cytokine activity.

The present invention provides methods for regulating the shedding ofthe extracellular domain of at least one cytokine receptor, comprisingthe steps of: providing a recombinant vector comprising SEQ ID NO:1 inthe sense orientation, a first tissue containing one or more cellsexpressing at least one cytokine receptor, and a second tissuecomprising one or more cells capable of expressing the polypeptideencoded by the recombinant vector; delivering the vector to the cells ofthe second tissue in the presence of the first tissue, under conditionswhich result in regulation of shedding of the cytokine receptor(s) fromcells of the first tissue. In some preferred embodiments, the cytokinereceptor is selected from the group consisting of type-1 tumor necrosisfactor receptor, type I interleukin-1 cytokine receptor, type IIinterleukin-1 cytokine receptor, and interleukin-6 cytokine receptoralpha-chain gp80. In alternative preferred embodiments, the delivery ofthe vector to the second tissue comprises a means of intracellulardelivery selected from the group consisting of direct nucleic acidadministration, liposome administration, viral vector delivery, and exvivo gene delivery followed by transplantation.

In other embodiments, the present invention provides compositions andmethods suitable for regulating TNFR1 ectodomain shedding byoverexpressing or suppressing the activity of the ARTS-1 polypeptide. Insome of these embodiments, TNFR1 ectodomain shedding is regulated by theintracellular delivery of a vector which results in overexpression ofthe ARTS-1 polypeptide (e.g., SEQ ID NO:2). In another embodiment, TNFR1ectodomain shedding is regulated by delivering purified ARTS-1polypeptide (e.g., SEQ ID NO:2) to tissues.

The present invention also provides methods for regulating the sheddingof the extracellular domain of at least one cytokine receptor,comprising the steps of: providing a recombinant vector comprising atleast a transcribeable portion of the nucleic acid of SEQ ID NO:1 in anantisense orientation, a first tissue comprising one or more cellsexpressing at least one cytokine receptor, and a second tissuecomprising one or more cells expressing the endogenous polypeptide ofSEQ ID NO:2, and one or more cells capable of transcribing the antisensenucleic acid; and delivering the vector to the second tissue in thepresence of the first tissue, under conditions that result in regulationof shedding of the cytokine receptor(s) from the cells of the firsttissue. In preferred embodiments, the cytokine receptor is selected fromthe group consisting of type-1 tumor necrosis factor receptor, type Iinterleukin-1 cytokine receptor, type II interleukin-1 cytokinereceptor, and interleukin-6 cytokine receptor alpha-chain gp80. Inalternative preferred embodiments, the delivery of the vector to thesecond tissue comprises a means of intracellular delivery selected fromthe group consisting of direct nucleic acid administration, liposomeadministration, viral vector delivery, and ex vivo gene deliveryfollowed by transplantation.

The present invention further provides methods for regulating theshedding of the extracellular domain of at least one cytokine receptor,comprising the steps of: providing a polypeptide having the amino acidsequence set forth in SEQ ID NO:2, and a tissue comprising one or morecells expressing at least one cytokine receptor on their plasma membraneextracellular surface; and delivering the polypeptide to the tissueunder conditions such that the polypeptide regulates the shedding of thecytokine receptor(s) from the surface of the cells of the tissue. Insome preferred embodiments, the cytokine receptor is selected from thegroup consisting of type-1 tumor necrosis factor receptor, type Iinterleukin-1 cytokine receptor, type II interleukin-1 cytokinereceptor, and interleukin-6 cytokine receptor alpha-chain gp80. Inalternative preferred embodiments, the delivery of the polypeptide tothe tissue comprises a means of delivery selected from the groupconsisting of oral administration, intra-arterial injection, intravenousinjection, intramuscular injection, intraperitoneal injection,subcutaneous injection, suppository, local surgical administration,systemic surgical administration, catheter, and any combination of thesemeans of delivery.

The present invention further provides methods for regulating theshedding of the extracellular domain of at least one cytokine receptor,providing an antibody raised against the ARTS-1 polypeptide, and atissue comprising one or more cells expressing at least one cytokinereceptor and the endogenous polypeptide of SEQ ID NO:2; and deliveringthe antibody to the tissue under conditions such that the antibodyregulates the shedding of the cytokine receptor(s) from the surface ofthe cells of the tissue. In some preferred embodiments, the cytokinereceptor is selected from the group consisting of type-1 tumor necrosisfactor receptor, type I interleukin-1 cytokine receptor, type IIinterleukin-1 cytokine receptor, and interleukin-6 cytokine receptoralpha-chain gp80. In alternative preferred embodiments, the means ofdelivery is selected from the group consisting of oral administration,intra-arterial injection, intravenous injection, intramuscularinjection, intraperitoneal injection, subcutaneous injection,suppository, local surgical administration, systemic surgicaladministration, catheter, and any combination of these means ofdelivery.

In view of the overlapping activities between TNF and otherproinflammatory cytokines, the present invention also providescompositions and methods suitable for regulating the shedding of othercytokine receptors in addition to TNFR1, including, but not limited toIL-1 and IL-6 cytokine receptors.

In some particularly preferred embodiments, the present inventionprovides compositions and methods to treat subjects displayingpathology, as well as subjects suspected of displaying or at risk ofdisplaying pathology resulting from abnormal cytokine activity. Thecompositions provided for use in the most preferred embodiment includevectors capable of expressing the ARTS-1 polypeptide, vectors capable oftranscribing at least a portion of the ARTS-1 gene in an antisenseorientation, the ARTS-1 polypeptide set forth in SEQ ID NO:2, andantibodies raised against at least a portion of the ARTS-1 polypeptide.

The present invention also provides methods for treating a subject,comprising the steps of: providing a composition selected from the groupconsisting of a recombinant vector comprising at least a portion of SEQID NO:1 in the sense orientation, a recombinant vector comprising atleast a portion of SEQ ID NO:1 in the antisense orientation, at least aportion of the ARTS-1 polypeptide, at least a portion of SEQ ID NO:2,and antibody directed against at least a portion of the ARTS-1polypeptide, as well as a subject, and a means of delivery of thecomposition to at least one tissue of the subject; and delivering thecomposition to the subject using the means of delivery. In preferredembodiments, the subject is selected from the group consisting of asubject displaying pathology resulting from abnormal cytokine activity,a subject suspected of displaying pathology resulting from abnormalcytokine activity, and a subject at risk of displaying pathologyresulting from abnormal cytokine activity. In some preferredembodiments, the cytokine activity is mediated by a cytokine selectedfrom the group consisting of tumor necrosis factor α, interleukin-1alpha, interleukin-1 beta, and interleukin-6. In some particularlypreferred embodiments, the subject is a human. In alternative preferredembodiments, the means of delivery is selected from the group consistingof oral administration, intra-arterial injection, intravenous injection,intramuscular injection, intraperitoneal injection, subcutaneousinjection, suppository, local surgical administration, systemic surgicaladministration, catheter, and any combination of these means ofdelivery. In further preferred embodiments, the means of delivery isfurther selected from the group consisting of direct nucleic acidadministration, liposome administration, viral vector delivery, and exvivo gene delivery followed by transplantation.

In other embodiments, the present invention provides ARTS-1 markers,including those selected from the group consisting of the ARTS-1 mRNAtranscript and the ARTS-1 polypeptide. Furthermore, the invention alsoprovides compositions and means for detecting the ARTS-1 mRNA andpolypeptide markers. In preferred embodiments, these compositions areselected from the group consisting of nucleic acid complementary to theARTS-1 mRNA, and antibodies specific for at least a portion of theARTS-1 polypeptide.

The present invention also provides means for detecting an ARTS-1 mRNAin a sample, wherein the means comprises at least a portion of thenucleic acid of SEQ ID NO:1 complementary to at least a portion ofARTS-1 mRNA, and further wherein the nucleic acid is a probe. Inalternative embodiments, the means comprises Northern blotting. Inpreferred embodiments, the sample is a tissue sample from a subject. Inadditional preferred embodiments, the methods provide means fordetecting an ARTS-1 polypeptide in a sample, wherein the means comprisesan antibody directed against at least a portion of the ARTS-1polypeptide. In some preferred embodiments, the means comprises Westernimmunoblotting, while in alternative preferred embodiments, the meanscomprises an enzyme-linked immunosorbent assay. In alternative preferredembodiments, the sample is a tissue sample from a subject.

The present invention also provides diagnostic kits comprising a meansto measure ARTS-1 expression, wherein the means comprises at least aportion of SEQ ID NO:1 that is complementary to at least a portion ofARTS-1 mRNA, and further wherein the nucleic acid is a probe. Inalternative embodiments, the diagnostic kits of the present inventionprovides means for detecting an ARTS-1 polypeptide in a sample, whereinthe means comprises antibody directed against at least a portion of theARTS-1 polypeptide. In some preferred embodiments, the means comprisesWestern immunoblotting, while in alternative preferred embodiments, themeans comprises an enzyme-linked immunosorbent assay.

The present invention further provides compositions and methods for thescreening for drugs with the ability to regulate ARTS-1 peptidaseactivity and cytokine receptor shedding regulatory activity. In someembodiments, the present invention provides methods for drug screeningto identify drugs having the ability to regulate ARTS-1 expressioncomprising the steps of: providing a drug, cultured cells, and a meansto measure ARTS-1 expression, wherein the means comprises at least aportion of SEQ ID NO:1 that is complementary to at least a portion ofARTS-1 mRNA, and further wherein the nucleic acid is a probe; exposingthe cells to the drug; and using the means to measure ARTS-1 expression.In alternative embodiments, the diagnostic kits of the present inventionprovide means for detecting an ARTS-1 polypeptide in a sample, whereinthe means comprises an antibody directed against at least a portion ofthe ARTS-1 polypeptide. In some preferred embodiments, the culturedcells are human NCI-H292 pulmonary mucoepidermoid carcinoma cells.

The present invention further provides methods for drug screening toidentify drugs capable of regulating the peptidase activity of ARTS-1,comprising the steps of: providing purified ARTS-1 polypeptide, an aminoacid p-nitroaniline, a means to measure amino acid p-nitroanilinecleavage, and a drug; exposing the purified ARTS-1 polypeptide to theamino acid p-nitroaniline in the absence and presence of the drug; andmeasuring amino acid p-nitroaniline cleavage in the absence and presenceof the drug. In some preferred embodiments, the purified ARTS-1polypeptide comprises glutathione-S-transferase. In alternativepreferred embodiments, the amino acid p-nitroaniline is selected fromthe group consisting of isoleucine p-nitroanilide, phenylalaninep-nitroanilide and glycine p-nitroanilide. In still further preferredembodiments, the means to measure amino acid p-nitroaniline cleavagecomprises measuring absorbance at 380 nm.

The present invention also provides methods for drug screening toidentify drugs capable of regulating the shedding of a cytokinereceptor, comprising the steps of: providing cultured cells expressingat least one cytokine receptor, a means to quantitate the concentrationof the soluble form of the cytokine receptor(s) in the supernatants ofthe cultured cells, and a drug; culturing the cells in the absence andpresence of the drug; quantitating the concentration of the soluble formof the cytokine receptor(s) in the supernatants of the cultured cells;and comparing the concentrations of the soluble cytokine receptor(s) inthe supernatants of the cell cultures in the absence and presence ofdrug. In some preferred embodiments, the cytokine receptor is selectedfrom the group consisting of type-1 tumor necrosis factor receptor, typeII interleukin-1 cytokine receptor, and interleukin-6 cytokine receptoralpha-chain gp80. In alternative preferred embodiments, the means toquantitate the concentration of the soluble form of a cytokine receptorcomprises an enzyme-linked immunosorbent assay. In further embodiments,the cultured cells are cultured human NCI-H292 pulmonary mucoepidermoidcarcinoma cells.

DESCRIPTION OF THE FIGURES

FIG. 1 presents the ARTS-1 transcription unit and open reading frametranslation.

FIG. 2 provides a schematic representation of the ARTS-1 protein,indicating domains of homology with the aminopeptidase family ofgluzincin zinc metalloproteases.

FIG. 3 provides a Northern blot analysis of multiple human tissues usinga ³²p-labelled ARTS-1 cDNA probe. The upper panel shows the blot probedwith the ARTS-1 cDNA probe. The lower panel shows the same blotfollowing stripping and rehybridization to a probe specific for thehuman glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene as areference for RNA loading normalization.

FIG. 4 provides Western immunoblots using pre-immune and polyclonalanti-ARTS-1 antisera in conjunction with crude whole cell homogenates,and membrane and cytosolic fractions made from cultured NCI-H292 cells.The top panel provides a Western immunoblot using the immune sera. Thenext panel provides a Western immunoblot using the pre-immune sera. Thebottom two panel provide results from Western immunoblot competitionexperiments using bovine serum albumin or ARTS-1 cognate peptide,respectively.

FIG. 5 provides Western immunoblots using polyclonal anti-ARTS-1antisera and membrane and cytosolic fractions made from primary cellsand cell lines. Panel A provides a Western immunoblot using humanbronchial brush cells collected from human subjects. Panel B provides aWestern immunoblot using the NCI-H292, BEAS-2B, BET-1A and A549 culturedcell lines. Panel C provides a Western immunoblot using primary culturesof normal human bronchial epithelial cells (NHBE), human umbilical veinendothelial cells (HUVEC) and human fibroblasts.

FIG. 6 provides results of an analysis of recombinant GST-ARTS-1 fusionprotein purification and analysis of the aminopeptidase activity of thisprotein following purification. Panel A provides a Coomassie stained gelof protein samples obtained during the production and purification ofthe GST-ARTS-1 fusion protein. Panel B provides an FPLC elution profileof the purified GST-ARTS-1 fusion protein. Panel C provides the resultsof an assay of phenylalanine p-nitroaniline substrate aminopeptidaseactivity of the FPLC eluted fractions.

FIG. 7 provides a Western immunoblot using anti-ARTS-1 polyclonalantiserum as the primary antibody. Samples analyzed in the blot are frommembrane protein fractions derived from stably transfected NCI-H292cells which were untransfected (WT), control transfected (Mock), ARTS-1sense-overexpressing (ARTS-1) or ARTS-1 anti-sense expressing (AS). Twoindependent clones each from the ARTS-1 and AS cell lines were analyzed.

FIG. 8 provides results of an ELISA to determine the levels of sTNFR1resulting from TNFR1 ectodomain shedding in cell culture supernatantsfrom cultures of the same cell lines as indicated for FIG. 7.

FIG. 9 provides a graph depicting the ability of ARTS-1 overexpressionto potentiate the cleavage and shedding of TNFR ectodomain from thesurface of NCI-H292 cells in response to PMA stimulation, as determinedby an ELISA measuring sTNFR1 in the cell culture supernatants.

FIG. 10 provides a histogram showing the results of an ELISA analysis.The ELISA determined the levels of sTNFR1 (a measure of TNFR1 ectodomainshedding) in cell culture supernatants for stably transfected NCI-H292cell lines expressing various ARTS-1 mutants.

FIG. 11 provides a Western immunoblot using an anti-TNFR1 antibody asthe primary antibody to detect membrane bound TNFR1. Samples analyzed inthe blot include membrane protein fractions derived from stablytransfected NCI-H292 cells which were untransfected (WT), controltransfected (Mock), ARTS-1 sense-overexpressing (ARTS-1), or ARTS-1anti-sense expressing (AS). Two independent clones of each cell linewere analyzed.

FIG. 12 provides two Western immunoblots following two in vivoimmunoprecipitation experiments using membrane protein fractionsisolated from cultured NCI-H292 cells. In the top panel, an anti-TNFR1antibody was used in the immunoprecipitation step (indicated as “IP”),and anti-ARTS-1 antiserum was used as the primary antibody in theimmunoblotting (indicated as “IB”). Conversely, in the lower panel, theanti-ARTS-1 antiserum was used in the immunoprecipitation, while theanti-TNFR1 antibody was used as the primary antibody in theimmunoblotting.

FIG. 13 provides a Western immunoblot following an in vivoimmunoprecipitation experiment using an anti-TNFR1 monoclonal antibodyfor the immunoprecipitation and anti-ARTS-1 antiserum as the primaryantibody in the blot. The immunoprecipitations used cell membraneprotein fractions derived from stably transfected NCI-H292 cellsoverexpressing ARTS-1 (ARTS-1), expressing an anti-sense ARTS-1 message(AS), as well as control-transfected (Mock) and non-transfected (WT)cell lines.

FIG. 14 provides results of two drug screening assays. Panel A providesa Western immunoblot using anti-ARTS-1 polyclonal antiserum as theprimary antibody, tested with membrane protein fractions from NCI-H292cells following exposure to 4b-phorbol 12-myristate 13-acetate (PMA)(over a time course). Panel B provides the results of an ELISA todetermine the levels of sTNFR1 in cell culture supernatants fromNCI-H292 cell cultures following exposure PMA, over time.

DEFINITIONS

To facilitate an understanding of the present invention, a number ofterms and phrases are defined or clarified below:

The terms “peptide,” “polypeptide” and “protein” all refer to a primarysequence of amino acids that are joined by covalent “peptide linkages.”In general, a peptide consists of a few amino acids, typically from 2–25amino acids, and is shorter than a protein. Polypeptides may encompasseither peptides or proteins. Where “amino acid sequence” is recitedherein to refer to an amino acid sequence of a naturally occurringprotein molecule, “amino acid sequence” and like terms, such as“polypeptide” or “protein” are not meant to limit the amino acidsequence to the complete, native amino acid sequence associated with therecited protein molecule.

As used herein, the term “nucleic acid” refers to any sequence of thebases adenine, thymine, cytosine and guanine, and various analogs ofthese bases. A nucleic acid is characterized by a specific nucleotidesequence (i.e., the sequence of the bases and base analogs in themolecule). A “nucleic acid” is not limited to DNA or RNA, and is notlimited in any way by the size of the molecule. A nucleic acid may bedouble stranded or single stranded. The term “nucleic acid” encompassessequences that include any of the bases adenine, thymine, guanine andcytosine, as well as known analogs of these bases, including but notlimited to, 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl) uracil,5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethylaminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

As used herein, the term “oligonucleotide,” refers to a short length ofsingle-stranded polynucleotide chain. Oligonucleotides are typicallyless than 100 residues long (e.g., between 15 and 50), however, as usedherein, the term is also intended to encompass longer polynucleotidechains. Oligonucleotides are often referred to by their length. Forexample a 24 residue oligonucleotide is referred to as a “24-mer.”Oligonucleotides can form secondary and tertiary structures byself-hybridizing or by hybridizing to other polynucleotides. Suchstructures can include, but are not limited to, duplexes, hairpins,cruciforms, bends, and triplexes.

As used herein, “recombinant nucleic acid,” “recombinant gene” or“recombinant DNA molecule” indicate that the nucleotide sequence orarrangement of its parts is not a native configuration, and has beenmanipulated by molecular biological techniques. The term implies thatthe DNA molecule is comprised of segments of DNA that have beenartificially joined together. Protocols and reagents to manipulatenucleic acids are common and routine in the art (See e.g., Maniatis etal.(eds.), Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY, [1982]; Sambrook et al. (eds.), Molecular Cloning:A Laboratory Manual, Second Edition, Volumes 1–3, Cold Spring HarborLaboratory Press, NY, [1989]; and Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, Vol. 1–4, John Wiley & Sons, Inc., NewYork [1994]).

Similarly, a “recombinant protein” or “recombinant polypeptide” refersto a protein molecule that is expressed from a recombinant DNA molecule.Use of these terms indicates that the primary amino acid sequence,arrangement of its domains or nucleic acid elements which control itsexpression are not native, and have been manipulated by molecularbiology techniques. As indicated above, techniques to manipulaterecombinant proteins are also common and routine in the art.

The terms “exogenous” and “heterologous” are sometimes usedinterchangeably with “recombinant.” An “exogenous nucleic acid,”“exogenous gene” and “exogenous protein” indicate a nucleic acid, geneor protein, respectively, that has come from a source other than itsnative source, and has been artificially supplied to the biologicalsystem. In contrast, the terms “endogenous protein,” “native protein,”“endogenous gene,” and “native gene” refer to a protein or gene that isnative to the biological system, species or chromosome under study. A“native” or “endogenous” polypeptide does not contain amino acidresidues encoded by recombinant vector sequences; that is, the nativeprotein contains only those amino acids found in the polypeptide orprotein as it occurs in nature. A “native” polypeptide may be producedby recombinant means or may be isolated from a naturally occurringsource. Similarly, a “native” or “endogenous” gene is a gene that doesnot contain nucleic acid elements encoded by sources other than thechromosome on which it is normally found in nature.

As used herein, the term “portion” when in reference to a protein (as in“a portion of a given protein”) refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid.

Nucleic acid molecules (e.g., DNA or RNA) are said to have “5′ ends” and“3′ ends” because mononucleotides are reacted to make oligonucleotidesor polynucleotides in a manner such that the 5′ phosphate of onemononucleotide pentose ring is attached to the 3′ oxygen of its neighborin one direction via a phosphodiester linkage. Therefore, an end of anoligonucleotide or polynucleotide is referred to as the “5′ end” if its5′ phosphate is not linked to the 3′ oxygen of a mononucleotide pentosering and as the “3′ end” if its 3′ oxygen is not linked to a 5′phosphate of a subsequent mononucleotide pentose ring. As used herein, anucleic acid sequence, even if internal to a larger oligonucleotide orpolynucleotide, also may be said to have 5′ and 3′ ends. In either alinear or circular DNA molecule, discrete elements are referred to asbeing “upstream” or 5′ of the “downstream” or 3′ elements. Thisterminology reflects the fact that transcription proceeds in a 5′ to 3′fashion along the DNA strand. The promoter and enhancer elements thatdirect transcription of a linked gene are generally located 5′ orupstream of the coding region. However, enhancer elements can exerttheir effect even when located 3′ of the promoter element or the codingregion. Transcription termination and polyadenylation signals arelocated 3′ or downstream of the coding region.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence comprisedof parts, that when appropriately combined in either a native orrecombinant manner, provide some product or function. Genes may or maynot comprise coding sequences necessary for the production of apolypeptide. Examples of genes which do not encode polypeptide sequencesinclude ribosomal RNA genes (rRNA) and transfer RNA (tRNA) genes. Genescan encode a polypeptide or any portion of a polypeptide within thegene's “coding region” or “open reading frame.” The polypeptide producedby the open reading frame of a gene may or may not display functionalactivity or properties of the full-length polypeptide (e.g., enzymaticactivity, ligand binding, signal transduction, etc.).

In addition to the coding region of the nucleic acid, the term “gene”also encompasses the transcribed nucleotide sequences of the full-lengthmRNA adjacent to the 5′ and 3′ ends of the coding region. Thesenoncoding regions are variable in size, and typically extend fordistances up to or exceeding 1 kb on both the 5′ and 3′ ends of thecoding region. The sequences that are located 5′ and 3′ of the codingregion and are contained on the mRNA are referred to as 5′ and 3′untranslated sequences (5′ UT and 3′ UT). Both the 5′ and 3′ UT mayserve regulatory roles, including translation initiation,post-transcriptional cleavage and polyadenylation. The term “gene”encompasses mRNA, cDNA and genomic forms of a gene.

It is contemplated that the genomic form or genomic clone of a gene maycontain the sequences of the transcribed mRNA, as well as othernon-coding sequences which lie outside of the mRNA. The regulatoryregions which lie outside the mRNA transcription unit are sometimescalled “5′ or 3′ flanking sequences.” A functional genomic form of agene must contain regulatory elements necessary for the regulation oftranscription. The term “promoter/enhancer region” is usually used todescribe this DNA region, typically but not necessarily 5′ of the siteof transcription initiation, sufficient to confer appropriatetranscriptional regulation. The word “promoter” alone is sometimes usedsynonymously with “promoter/enhancer.” A promoter may be constitutivelyactive, or alternatively, conditionally active, where transcription isinitiated only under certain physiological conditions or in the presenceof certain drugs. The 3′ flanking region may contain additionalsequences which regulate transcription, especially the termination oftranscription. “Introns” or “intervening regions” or “interveningsequences” are segments of a gene which are contained in the primarytranscript (i.e., hetero-nuclear RNA, or hnRNA), but are spliced out toyield the processed mRNA form. Introns may contain transcriptionalregulatory elements such as enhancers. The mRNA produced from thegenomic copy of a gene is translated in the presence of ribosomes toyield the primary amino acid sequence of the polypeptide.

As used herein, the term “regulatory element” refers to a geneticelement which controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element that enablesthe initiation of transcription of an operably linked coding region.Other regulatory elements are splicing signals, polyadenylation signals,termination signals, etc.

Transcriptional control signals in eukaryotes comprise “promoter” and“enhancer” elements. Promoters and enhancers consist of short arrays ofDNA sequences that interact specifically with cellular proteins involvedin transcription (Maniatis et al., Science 236:1237 [1987]). Promoterand enhancer elements have been isolated from a variety of eukaryoticsources including genes in yeast, insect and mammalian cells, as well asviruses. Analogous control elements (i.e., promoters and enhancers) arealso found in prokaryotes. The selection of a particular promoter andenhancer to be operably linked in a recombinant gene depends on whatcell type is to be used to express the protein of interest. Someeukaryotic promoters and enhancers have a broad host range while othersare functional only in a limited subset of cell types (for review see,Voss et al., Trends Biochem. Sci., 11:287 [1986] and Maniatis et al.,Science 236:1237 [1987]). For example, the SV40 early gene enhancer isvery active in a wide variety of mammalian cell types (Dijkema et al.,EMBO J., 4:761 [1985]). Two other examples of promoter/enhancer elementsactive in a broad range of mammalian cell types are those from the humanelongation factor 1α gene (Uetsuki et al., J. Biol. Chem., 264:5791[1989]; Kim et al., Gene 91:217 [1990]; Mizushima and Nagata, Nuc.Acids. Res., 18:5322 [1990]), the long terminal repeats of the Roussarcoma virus (Gorman et al., Proc. Natl. Acad. Sci. USA 79:6777[1982]), and human cytomegalovirus (Boshart et al., Cell 41:521 [1985]).Some promoter elements serve to direct gene expression in atissue-specific manner.

As used herein, the term “promoter/enhancer” denotes a segment of DNAwhich contains sequences capable of providing both promoter and enhancerfunctions (i.e., the functions provided by a promoter element and anenhancer element). For example, the long terminal repeats ofretroviruses contain both promoter and enhancer functions. Thepromoter/enhancer may be “endogenous,” or “exogenous,” or“heterologous.” An “endogenous” promoter/enhancer is one which isnaturally linked with a given gene in the genome. An “exogenous” or“heterologous” promoter/enhancer is one placed in juxtaposition to agene by means of genetic manipulation (i.e., molecular biologicaltechniques such as cloning and recombination) such that transcription ofthe gene is controlled by the linked promoter/enhancer.

The presence of “splicing signals” on an expression vector often resultsin higher levels of expression of the recombinant transcript. Splicingsignals mediate the removal of introns from the primary RNA transcriptand consist of a splice donor and acceptor site (See e.g., Sambrook etal., Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, New York [1989], pp. 16.7–16.8). A commonlyused splice donor and acceptor site is the splice junction from the 16SRNA of SV40.

Efficient expression of recombinant DNA sequences in eukaryotic cellsrequires the presence of signals directing the efficient termination andpolyadenylation of the resulting transcript. Transcription terminationsignals are generally found downstream of the polyadenylation signal andare a few hundred nucleotides in length. The term “poly A site” or “polyA sequence” as used herein denotes a nucleic acid sequence that directsboth the termination and polyadenylation of the nascent RNA transcript.Efficient polyadenylation of the recombinant transcript is desirable astranscripts lacking a poly A tail are unstable and are rapidly degraded.The poly A signal utilized in an expression vector may be “heterologous”or “endogenous.” An endogenous poly A signal is one that is foundnaturally at the 3′ end of the coding region of a given gene in thegenome. A heterologous poly A signal is one that is isolated from onegene and placed 3′ of another gene. A commonly used heterologous poly Asignal is the SV40 poly A signal. The SV40 poly A signal is contained ona 237 bp BamHI/BclI restriction fragment and directs both terminationand polyadenylation (See e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press,New York [1989], pp. 16.6–16.7).

The terms “in operable combination,” “in operable order,” “operablylinked” and similar phrases when used in reference to nucleic acidherein are used to refer to the linkage of nucleic acid sequences insuch a manner that a nucleic acid molecule capable of directing thetranscription of a given gene and/or the synthesis of a desired proteinmolecule is produced. The term also refers to the linkage of amino acidsequences in such a manner so that a functional protein is produced.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene,” “polynucleotide having a nucleotide sequenceencoding a gene,” and similar phrases are meant to indicate a nucleicacid sequence comprising the coding region of a gene (i.e., the nucleicacid sequence which encodes a gene product). The coding region may bepresent in either a cDNA, genomic DNA or RNA form. When present in a DNAform, the oligonucleotide, polynucleotide or nucleic acid may besingle-stranded (i.e., the sense strand or the antisense strand) ordouble-stranded. Suitable control elements such as enhancers/promoters,splice junctions, polyadenylation signals, etc. may be placed in closeproximity to the coding region of the gene if needed to permit properinitiation of transcription and/or correct processing of the primary RNAtranscript. Alternatively, the coding region utilized in the expressionvectors of the present invention may contain endogenousenhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” and similar phrases refer to theorder or sequence of deoxyribonucleotides along a strand ofdeoxyribonucleic acid. The order of these deoxyribonucleotidesdetermines the order of amino acids along the polypeptide (e.g.,protein) chain. The DNA sequence thus codes for the amino acid sequence.

As used herein, the term “gene expression” refers to the process ofconverting genetic information encoded in a gene into RNA (e.g., mRNA,rRNA, tRNA, or snRNA) through “transcription” of the gene (i.e., via theenzymatic action of an RNA polymerase), and for protein encoding genes,into protein through “translation” of the mRNA. Gene expression can beregulated at many stages. “Up-regulation” or “activation” refers toregulation that increases the production of gene expression products(i.e., RNA or protein), while “down-regulation” or “repression” refersto regulation that decreases mRNA or protein production. Molecules(e.g., transcription factors) that are involved in up-regulation ordown-regulation are often called “activators” and “repressors,”respectively.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the rules of antiparallel base-pairing. For example, thesequence 5′-C-T-A-G-T-3′ is complementary to the sequence5′-A-C-T-A-G-3′. Complementarity may be “partial,” in which only some ofthe nucleic acids' bases are matched according to antiparallel basepairing rules. Also, there may be “complete” or “total” complementaritybetween two nucleic acids. The degree of complementarity between nucleicacid strands has significant effects on the efficiency and strength ofhybridization between nucleic acid strands. This is of particularimportance in polymerase chain reaction (PCR) amplification reactions,as well as detection methods that depend upon binding between nucleicacids. As used herein, the terms “antiparallel complementarity” and“complementarity” are synonymous.

As used herein, the term “antisense” is used in reference to any nucleicacid which is antiparallel to and complementary to another nucleic acid.Antisense DNA or RNA may be produced by any method. For example, a cDNAor a portion of a cDNA may be subcloned into an expression vectorcontaining a promoter which permits transcription either in vitro or invivo. The cDNA or a portion of the cDNA is subcloned in such a way thatit is in the reverse orientation relative to the direction oftranscription of the cDNA in its native chromosome. Transcription ofthis antisense cDNA produces an RNA transcript that is complementary andantiparallel to the native mRNA. The mechanism by which an antisensenucleic acid produces effects in a biological system is unclear,however, likely involves the formation of a duplex with itscomplementary nucleic acid within either the nucleus or cytoplasm of acell. These duplexes are theorized to block transcription of the nativemRNA or prevent its translation. Using antisense techniques, an“artificial knockout” mutant may be reproduced in an animal or animalcell line. The term “antisense strand” is used in reference to a nucleicacid strand that is complementary to the “sense” strand. The designation(−) (i.e., “negative”) is sometimes used in reference to the antisensestrand, with the designation (+) (i.e., “positive”) sometimes used inreference to the sense strand.

The following definitions are the commonly accepted definitions of theterms “identity,” “similarity” and “homology.” Percent identity is ameasure of strict amino acid conservation. Percent similarity is ameasure of amino acid conservation which incorporates both strictlyconserved amino acids, as well as “conservative” amino acidsubstitutions, where one amino acid is substituted for a different aminoacid having similar chemical properties (i.e. a “conservative”substitution). The term “homology” can pertain to either proteins ornucleic acids. Two proteins can be described as “homologous” or“non-homologous,” but the degree of amino acid conservation isquantitated by percent identity and percent similarity. Nucleic acidconservation is measured by the strict conservation of the basesadenine, thymine, guanine and cytosine in the primary nucleotidesequence. When describing nucleic acid conservation, conservation of thenucleic acid primary sequence is sometimes expressed as percenthomology. In the same nucleic acid, one region may show a highpercentage of nucleotide sequence conservation, while a different regioncan show no or poor conservation. Nucleotide sequence conservation cannot be inferred from an amino acid similarity score. Two proteins mayshow domains that in one region are homologous, while other regions ofthe same protein are clearly non-homologous.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization can bedemonstrated using a variety of hybridization assays (Southern blot,Northern Blot, slot blot, phage plaque hybridization, and othertechniques). These protocols are common in the art (See e.g., Sambrooket al. (eds.), Molecular Cloning: A Laboratory Manual, Second Edition,Volumes 1–3, Cold Spring Harbor Laboratory Press, NY, [1989]; Ausubel etal. (eds.), Current Protocols in Molecular Biology, Vol. 1–4, John Wiley& Sons, Inc., New York [1994]). Hybridization is the process of onenucleic acid pairing with an antiparallel counterpart which may or maynot have 100% complementarity. Two nucleic acids which contain 100%antiparallel complementarity will show strong hybridization. Twoantiparallel nucleic acids which contain no antiparallel complementarity(generally considered to be less than 30%) will not hybridize. Twonucleic acids which contain between 31–99% complementarity will show anintermediate level of hybridization. A single molecule that containspairing of complementary nucleic acids within its structure is said tobe “self-hybridized.”

As used herein, the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acids hybridize.“Low or weak stringency” conditions are reaction conditions which favorthe complementary base pairing and annealing of two nucleic acids. “Highstringency” conditions are those conditions which are less optimal forcomplementary base pairing and annealing. The art knows well thatnumerous variables affect the strength of hybridization, including thelength and nature of the probe and target (DNA, RNA, base composition,present in solution or immobilized, the degree of complementary betweenthe nucleic acids, the T_(m) of the formed hybrid, and the G:C ratiowithin the nucleic acids). Conditions may be manipulated to define lowor high stringency conditions: factors such as the concentration ofsalts and other components in the hybridization solution (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)as well as temperature of the hybridization and/or wash steps.Conditions of “low” or “high” stringency are specific for the particularhybridization technique used.

During hybridization of two nucleic acids under high stringencyconditions, complementary base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences. Thus, conditions of “weak” or “low” stringency are oftenrequired with nucleic acids that are derived from organisms that aregenetically diverse, as the frequency of complementary sequences isusually less. As used herein, two nucleic acids which are able tohybridize under high stringency conditions are considered “substantiallyhomologous.”

Whether sequences are “substantially homologous” may be verified usinghybridization competition assays. For example, a “substantiallyhomologous” nucleotide sequence is one that at least partially inhibitsa completely complementary probe sequence from hybridizing to a targetnucleic acid under conditions of low stringency. This is not to say thatconditions of low stringency are such that non-specific binding ispermitted; low stringency conditions require that the binding of twosequences to one another be a specific (i.e., selective) interaction.The absence of non-specific binding may be verified by the use of asecond target that lacks even a partial degree of complementarity (e.g.,less than about 30% identity); in the absence of non-specific bindingthe probe will not hybridize to the second non-complementary target.When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of highstringency.

A gene may produce multiple RNA species that are generated bydifferential splicing of the primary RNA transcript. cDNAs that aresplice variants of the same gene contain regions of nucleotide sequenceidentity (100% homology), representing the presence of the same exon orportion of the same exon on both cDNAs, as well as regions of completenon-identity. Because the two cDNAs contain regions of sequence identitythey will both hybridize to a probe derived from the entire gene orportions of the gene containing sequences found on both cDNAs. As usedherein, the two splice variants are therefore substantially homologousto such a probe and to each other.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985]). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein, the term “amplifiable nucleic acid” is used in referenceto nucleic acids which may be amplified by any amplification method. Itis contemplated that “amplifiable nucleic acid” will usually comprise“template.” As used herein, the term “template” refers to nucleic acidoriginating from a sample that is to be used as a substrate for thegeneration of the amplified nucleic acid.

As used herein, the term “primer” refers to an oligonucleotide,typically but not necessarily produced synthetically, that is capable ofacting as a point of initiation of nucleic acid synthesis when placedunder conditions in which synthesis of a primer extension product thatis complementary to a nucleic acid strand is induced, (i.e., in thepresence of nucleotides, an inducing agent such as DNA polymerase, andat a suitable temperature and pH). The primer is preferably singlestranded for maximum efficiency in amplification, but may alternativelybe double stranded. If double stranded, the primer is first treated toseparate its strands before being used to prepare extension products.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer and theuse of the method.

As used herein, the term “probe” refers to an oligonucleotide (i.e., asequence of nucleotides), whether produced as a purified restrictiondigest or produced by synthetic means, recombinantly or byamplification, that is capable of hybridizing to another oligonucleotideof interest. A probe may be single-stranded or double-stranded. Probesare useful in the detection, identification and isolation of particulargene sequences. It is contemplated that in preferred embodiments, anyprobe used in the present invention will be labelled with any “reportermolecule,” so that it is detectable in any detection system, including,but not limited to enzyme (e.g., ELISA, as well as immunohistochemicalassays), fluorescent, radioactive, and luminescent systems. It is notintended that the present invention be limited to any particulardetection system or label.

As used herein, the term “polymerase chain reaction” (“PCR”) refers tothe method of K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202 and4,965,188, each of which is hereby incorporated by reference, whichdescribe a method for increasing the concentration of a segment of atarget sequence in a mixture of genomic DNA without cloning orpurification. This process for amplifying the target sequence consistsof introducing a large excess of two oligonucleotide primers to the DNAmixture containing the desired target sequence, followed by a precisesequence of thermal cycling in the presence of a thermostable DNApolymerase. The two primers are complementary to their respectivestrands of the double stranded target sequence. To effect amplification,the mixture is denatured and the primers then annealed to theircomplementary sequences within the target molecule. Following annealing,the primers are extended with a polymerase so as to form a new pair ofcomplementary strands. The steps of denaturation, primer annealing andpolymerase extension can be repeated many times (i.e., denaturation,annealing and extension constitute one “cycle”; there can be numerous“cycles”) to obtain a high concentration of an amplified segment of thedesired target sequence. The length of the amplified segment of thedesired target sequence is determined by the relative positions of theprimers with respect to each other, and therefore, this length is acontrollable parameter. By virtue of the repeating aspect of theprocess, the method is referred to as the “polymerase chain reaction”(hereinafter “PCR”). Because the desired amplified segments of thetarget sequence become the predominant sequences (in terms ofconcentration) in the mixture, they are said to be “PCR amplified”.

With PCR, it is possible to amplify a single copy of a specific targetsequence in genomic DNA to a level detectable by several differentmethodologies (e.g., hybridization with a labelled probe; incorporationof biotinylated primers followed by avidin-enzyme conjugate detection;incorporation of ³²P-labelled deoxynucleotide triphosphates, such asdCTP or dATP, into the amplified segment). In addition to genomic DNA,any oligonucleotide or polynucleotide sequence can be amplified with theappropriate set of primer molecules. In particular, the amplifiedsegments created by the PCR process are, themselves, efficient templatesfor subsequent PCR amplifications.

As used herein, the terms “PCR product,” “PCR fragment,” and“amplification product” refer to the resultant mixture of compoundsafter two or more cycles of the PCR steps of denaturation, annealing andextension are complete. These terms encompass the case where there hasbeen amplification of one or more segments of one or more targetsequences.

As used herein, the term “amplification reagents” refers to thosereagents (e.g., deoxyribonucleotide triphosphates, buffer, etc.), neededfor amplification except for primers, nucleic acid template and theamplification enzyme. Typically, amplification reagents along with otherreaction components are placed and contained in a reaction vessel (testtube, microwell, etc.).

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated nucleic acid,” “an isolated oligonucleotide” or “isolatedpolynucleotide” refers to a nucleic acid sequence that is identified andseparated from at least one contaminant nucleic acid with which it isordinarily associated in its natural source. Isolated nucleic acid ispresent in a form or setting that is different from the form or settingof that nucleic acid found in nature. In contrast, non-isolated nucleicacids are found in the state in which they exist in nature. For example,a given DNA sequence (e.g., a gene) is found on the host cell chromosomein proximity to neighboring genes; RNA sequences, such as a specificmRNA sequence encoding a specific protein, are found in the cell in amixture with numerous other mRNAs that encode a multitude of proteins.However, isolated nucleic acid encoding a given polypeptide includes, byway of example, such nucleic acid in cells ordinarily expressing thegiven protein where the nucleic acid is in a chromosomal locationdifferent from that of natural cells, or is otherwise flanked by adifferent nucleic acid sequence than that found in nature. The isolatednucleic acid, oligonucleotide, or polynucleotide may be present insingle-stranded or double-stranded form. When an isolated nucleic acid,oligonucleotide or polynucleotide is to be utilized to express aprotein, the oligonucleotide or polynucleotide will contain at a minimumthe sense or coding strand (i.e., the oligonucleotide or polynucleotidemay be single-stranded), but may contain both the sense and anti-sensestrands (i.e., the oligonucleotide or polynucleotide may bedouble-stranded).

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample. For example, antibodies are purified byremoval of contaminating non-immunoglobulin proteins; they are alsopurified by the removal of immunoglobulin that does not bind to thetarget molecule. The removal of non-immnunoglobulin proteins and/or theremoval of immunoglobulins that do not bind to the target moleculeresults in an increase in the percent of target-reactiveinmmunoglobulins in the sample. In another example, recombinantpolypeptides are expressed in bacterial host cells and the polypeptidesare purified by the removal of host cell proteins; the percent ofrecombinant polypeptides is thereby increased in the sample.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.” A vector“backbone” comprises those parts of the vector which mediate itsmaintenance and enable its intended use (e.g., the vector backbone maycontain sequences necessary for replication, genes imparting drug orantibiotic resistance, a multiple cloning site, and possibly operablylinked promoter/enhancer elements which enable the expression of acloned nucleic acid). The cloned nucleic acid (e.g., such as a cDNAcoding sequence, or an amplified PCR product) is inserted into thevector backbone using common molecular biology techniques. Vectors areoften derived from plasmids, bacteriophages, or plant or animal viruses.A “cloning vector” or “shuttle vector” or “subcloning vector” containoperably linked parts which facilitate subcloning steps (e.g., amultiple cloning site containing multiple restriction endonucleasesites). A “recombinant vector” indicates that the nucleotide sequence orarrangement of its parts is not a native configuration, and has beenmanipulated by molecular biological techniques. The term implies thatthe vector is comprised of segments of DNA that have been artificiallyjoined.

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and operably linkednucleic acid sequences necessary for the expression of the operablylinked coding sequence in a particular host organism (e.g., a bacterialexpression vector, a yeast expression vector or a mammalian expressionvector). Nucleic acid sequences necessary for expression in prokaryotestypically include a promoter, an operator (optional), and a ribosomebinding site, often along with other sequences. Eukaryotic cells utilizepromoters, enhancers, and termination and polyadenylation signals andother sequences which are different from those used by prokaryotes.

Eukaryotic expression vectors may also contain “viral replicons” or“viral origins of replication.” Viral replicons are viral DNA sequencesthat allow for the extrachromosomal replication of a vector in a hostcell expressing the appropriate replication factors. Some vectorsreplicate their nucleic acid to high copy numbers (e.g., vectors thatcontain either the SV40 or polyoma virus origin of replication replicateto high “copy number” (up to 10⁴ copies/cell) in cells that express theappropriate viral T antigen). Other vectors replicate their nucleic acidin low copy numbers (e.g., vectors that contain the replicons frombovine papillomavirus or Epstein-Barr virus replicate extrachromosomallyat “low copy number” (˜100 copies/cell)). The viral origins ofreplication listed above are not limiting, as the art is aware of otherorigins of replication that are commonly used in eukaryotic expressionvectors.

The term “transgene” as used herein refers to a foreign gene that isplaced into an organism by, for example, introducing the foreign geneinto newly fertilized eggs or early embryos. The term “foreign gene”refers to any nucleic acid (e.g., gene sequence) that is introduced intothe genome of an animal by experimental manipulations and may includegene sequences found in that animal so long as the introduced gene doesnot reside in the same location as does the naturally-occurring gene.

Embryonal cells at various developmental stages can be used to introducetransgenes for the production of transgenic, non-human animals.Different methods are used depending on the stage of development of theembryonic cell. The zygote is the best target for micro-injection. Inthe mouse, the male pronucleus reaches the size of approximately 20micrometers in diameter which allows reproducible injection of 1–2picoliters (pl) of DNA solution. The use of zygotes as a target for genetransfer has a major advantage in that in most cases the injected DNAwill be incorporated into the host genome before the first cleavage(Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438–4442 [1985]). As aconsequence, all cells of the transgenic non-human animal will carry theincorporated transgene. This will, in general, also be reflected in theefficient transmission of the transgene to offspring of the foundersince 50% of the germ cells will harbor the transgene. Micro-injectionof zygotes is the preferred method for incorporating transgenes inpracticing the invention. U.S. Pat. No. 4,873,191, herein incorporatedby reference, describes a method for the micro-injection of zygotes.

Retroviral infection can also be used to introduce transgenes into anon-human animal. The developing embryo can be cultured in vitro to theblastocyst stage. During this time, the blastomeres can be targets forretroviral infection (Jaenisch, Proc. Natl. Acad. Sci. USA 73:1260–1264[1976]). Efficient infection of the blastomeres is obtained by enzymatictreatment to remove the zona pellucida (See e.g., Hogan et al., inManipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. [1986]). The viral vector system used to introducethe transgene is typically a replication-defective retrovirus carryingthe transgene (Jahner et al., Proc. Natl. Acad Sci. USA 82:6927–693[1985]). Transfection is easily and efficiently obtained by culturingthe blastomeres on a monolayer of virus-producing cells (van der Putten,Proc. Natl. Acad. Sci. USA 82(18):6148–52 [1985]; and Stewart, et al.,EMBO J. 6:383–388 [1987]). Alternatively, infection can be performed ata later stage. Virus or virus-producing cells can be injected into theblastocoele (Jahner et al., Nature 298:623–628 [1982]). Most of thefounders will be mosaic for the transgene since incorporation occursonly in a subset of cells that form the transgenic animal. Further, thefounder may contain various retroviral insertions of the transgene atdifferent positions in the genome that generally will segregate in theoffspring. In addition, it is also possible to introduce transgenes intothe germline, albeit with low efficiency, by intrauterine retroviralinfection of the midgestation embryo (Jauner et al., Nature 298:623–628[1982]). Additional means of using retroviruses or retroviral vectors tocreate transgenic animals known to the art include, but are not limitedto, the micro-injection of retroviral particles or mitomycin C-treatedcells producing retrovirus into the perivitelline space of fertilizedeggs or early embryos (PCT International Application WO 90/08832 [1990],and Haskell and Bowen, Mol. Reprod. Dev., 40:386 [1995]).

A third type of target cell for transgene introduction is the embryonalstem (ES) cell. ES cells are obtained by culturing pre-implantationembryos in vitro under appropriate conditions (Evans et al., Nature292:154–156 [1981]; Bradley et al., Nature 309:255–258 [1984]; Gossleret al., Proc. Acad. Sci. USA 83:9065–9069 [1986]; and Robertson et al.,Nature 322:445–448 [1986]). Transgenes can be efficiently introducedinto the ES cells by DNA transfection using a variety of methods knownto the art including calcium phosphate co-precipitation, protoplast orspheroplast fusion, lipofection and DEAE-dextran-mediated transfection.Transgenes may also be introduced into ES cells by retrovirus-mediatedtransduction or by micro-injection. Such transfected ES cells canthereafter colonize an embryo following their introduction into theblastocoel of a blastocyst-stage embryo and contribute to the germ lineof the resulting chimeric animal (for review, see Jaenisch, Science240:1468–1474 [1988]). Prior to the introduction of transfected ES cellsinto the blastocoel, the transfected ES cells may be subjected tovarious selection protocols to enrich for ES cells that have integratedthe transgene, assuming that the transgene provides a means for suchselection. Alternatively, PCR may be used to screen for ES cells thathave integrated the transgene. This technique obviates the need forgrowth of the transfected ES cells under appropriate selectiveconditions prior to transfer into the blastocoel.

The terms “overexpression” and “overexpressing” and grammaticalequivalents are used in reference to levels of mRNA or protein where thelevel of expression of the mRNA or protein is higher than that typicallyobserved in a given tissue in a control or non-transgenic animal. Levelsof mRNA or protein are measured using any of a number of techniquesknown to those skilled in the art. For example, mRNA levels may beassayed using (but not limited to) a Northern blot analysis. Appropriatecontrols are included on the Northern blot to control for differences inthe amount of RNA loaded from each tissue analyzed (e.g., the amount of28S rRNA, an abundant RNA transcript present at essentially the sameamount in all tissues, present in each sample can be used as a means ofnormalizing or standardizing the mRNA-specific signal observed onNorthern blots). The amount of mRNA present in the band corresponding insize to the correctly spliced transgene RNA is quantified; other minorspecies of RNA which hybridize to the transgene probe are not consideredin the quatntification of the expression of the transgenic mRNA.

The term “transfection” as used herein refers to the introduction offoreign DNA into cells. Transfection may be accomplished by a variety ofmeans known to the art including calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,electroporation, microinjection, liposome fusion, lipofection,protoplast fusion, retroviral infection, and biolistics. Mammalian celltransfection-techniques are common in the art, and are described in manysources (See, e.g., Ausubel et al. (eds.), Current Protocols inMolecular Biology, Vol. 1–4, John Wiley & Son, Inc., New YorK [1994]).

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell whichcontains stably integrated foreign DNA within its own genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells which have taken up foreign DNAbut have failed to integrate this DNA.

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a eukaryotic cell, and mosttypically mammalian cells. The uptake of nucleic acids by cells isenhanced when the nucleic acid is presented as a calciumphosphate-nucleic acid co-precipitate. Various modifications of theoriginal technique of Graham and van der Eb (Graham and van der Eb,Virol., 52:456 [1973]) are known in which the conditions for thetransfection of a particular cell type has been optimized. The art iswell aware of these various methods.

The term “transformation” has multiple meanings, depending on its usage.In one sense, the term “transformation” is used to describe the processof introduction of foreign DNA into prokaryotic cells (i.e., bacterialcells), and most frequently E. coli strains. Bacterial celltransformation may be accomplished by a variety of means well known tothe art, including the preparation of “competent” bacteria by the use ofcalcium chloride, magnesium chloride or rubidium chloride, andelectroporation. When a plasmid is used as the transformation vector,the plasmid typically contains a gene conferring drug resistance, suchas the genes encoding ampicillin, tetracycline or kanamycin resistance.Bacterial transformation techniques are common in the art, and aredescribed in many sources (e.g., Cohen et al., Proc. Natl. Acad. Sci.USA 69: 2110–2114 [1972]; Hanahan, J. Mol. Biol., 166:557–580 [1983];Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, SecondEdition, Volumes 1–3, Cold Spring Harbor Laboratory Press, NY, [1989];Ausubel et al. (eds.), Current Protocols in Molecular Biology, Vol. 1–4,John Wiley & Sons, Inc., New York [1994]).

“Transformation” also describes the physiological process by which anormal eukaryotic cell acquires the phenotypic qualities of a malignantcell. Such properties can include the ability to grow in soft agar, theability to grow in nutrient poor conditions, rapid proliferation, andthe loss of contact inhibition. A eukaryotic cell which is “transformed”displays the properties of malignant cells. A eukaryotic cell mayacquire its transformed phenotype in vivo, or be artificiallytransformed in culture.

As used herein, the term “selectable marker” refers to the use of a genethat encodes an enzymatic activity that confers the ability to grow inmedium lacking what would otherwise be an essential nutrient (e.g., theHIS3 gene in yeast cells); in addition, a selectable marker may conferresistance to an antibiotic or drug upon the cell in which theselectable marker is expressed. Furthermore, selectable markers may be“dominant.” Dominant selectable markers encode an enzymatic activitythat can be detected in any eukaryotic cell line. Examples of dominantselectable markers include the bacterial aminoglycoside 3′phosphotransferase gene (i.e., the neo gene) that confers resistance tothe drug G-418 in mammalian cells, as well as the bacterial hygromycin Gphosphotransferase (hyg) gene that confers resistance to the antibiotichygromycin, and the bacterial xanthine-guanine phosphoribosyltransferase gene (i.e., the gpt gene) that confers the ability to growin the presence of mycophenolic acid. The use of non-dominant selectablemarkers must be in conjunction with a cell line that lacks the relevantenzyme activity. Examples of non-dominant selectable markers include thethymidine kinase (tk) gene (used in conjunction with tk⁻ cell lines),the CAD gene (used in conjunction with CAD-deficient cells) and themammalian hypoxanthine-guanine phosphoribosyl transferase (hprt) gene(used in conjunction with hprt⁻ cell lines). A review of the use ofselectable markers in mammalian cell lines is provided in Sambrook etal., Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press, New York (1989), at pp. 16.9–16.15.

As used herein in the context of protein purification, the terms “sourceculture,” “starting culture,” “starting material” or “culture” or thelike can include any of the following materials: culture supernatant,cultured eukaryotic or prokaryotic cells (e.g., animal cells orbacteria), crushed eukaryotic or prokaryotic cells, tissue removed froman organism, or the product of an in vitro translation or in vitrocoupled transcription/translation reaction. The cells of such a culturemay or may not contain recombinant nucleic acid.

As used herein, the term “cell culture” refers to any in vitro cultureof cells. Included within this term are continuous cell lines (e.g.,with an immortal phenotype), primary cell cultures, finite cell lines(e.g., non-transformed cells), and any other cell population maintainedin vitro.

The term “test compound” refers to any chemical entity, pharmaceutical,drug, composition, and the like that can be used to treat or prevent adisease, illness, sickness, or disorder of bodily function. Testcompounds comprise both known and potential therapeutic compounds. A“known therapeutic compound” refers to a therapeutic compound that hasbeen previously described. Particularly preferred known therapeutics arethose that have been shown (e.g., through animal trials or priorexperience with administration to humans) to be effective in treatmentor prevention of pathology.

As used herein, a “drug” can be any molecule of any composition,including protein, peptide, nucleic acid, organic molecule, inorganicmolecule, or combinations of molecules, biological or non-biological,which are capable of producing a physiological response. As used herein,a “drug” provides at least one beneficial response in the cure,mitigation, treatment or prevention of a disease or disorder. A compoundis considered a “drug candidate” if it is not yet known if that compoundwill provide at least one beneficial response in the cure, mitigation,treatment or prevention of a disease, disorder or condition. A “druglibrary” is a collection of molecules, where it may or may not be knownif one or multiple drugs in the library have therapeutic value.

As used herein, the terms “host,” “expression host,” and “transformant”refer to organisms and/or cells which harbor an exogenous DNA sequence(e.g., via transfection), an expression vector or vehicle, as well asorganisms and/or cells that are suitable for use in expressing arecombinant gene or protein. It is not intended that the presentinvention be limited to any particular type of cell or organism. Indeed,it is contemplated that any suitable organism and/or cell will find usein the present invention as a host.

As used herein, the term “subject” refers to any animal being examined,studied or treated. It is not intended that the present invention belimited to any particular type of subject. It is contemplated thatmultiple organisms will find use in the present invention as subjects.In some embodiments, humans are the preferred subject.

A subject displaying pathology resulting from abnormal cytokine activitymay display symptoms that include, but are not limited to, inflammation,cachexia, insulin resistance, overstimulation of interleukin-6 andgranulocyte/macrophage-colony stimulating factor (GM-CSF) secretion,enhanced cytotoxicity of polymorphonuclear neutrophils, prolongedexpression of cellular adhesion molecules, induction of procoagulantactivity on vascular endothelial cells, increased adherence ofneutrophils and lymphocytes, stimulation of the release of plateletactivating factor from macrophages, neutrophils and vascular endothelialcells, fever, malaise, and anorexia.

As used herein, a “disease” is a disruption of normal body function,generally where two of three criteria are met: 1) the etiological agentis known, 2) an identifiable group of symptoms appears, and 3) there areconsistent anatomical or physiological alterations. Examples include,but are not limited to rheumatoid arthritis, inflammatory bowel diseaseand graft-versus-host disease. A disorder is, in general, a disruptionof some aspect of normal body function (e.g., rheumatoid arthritis andinflammatory bowel disease are immune disorders).

As used herein, the term “antigen” refers to any agent (e.g., anysubstance, compound, molecule [including macromolecules], or othermoiety), that is recognized by an antibody, while the term “immunogen”refers to any agent (e.g., any substance, compound, molecule [includingmacromolecules], or other moiety) that can elicit an immunologicalresponse in an individual. These terms may be used to refer to anindividual macromolecule or to a homogeneous or heterogeneous populationof antigenic macromolecules. It is intended that the terms antigen andimmunogen encompass protein molecules or at least one portion of aprotein molecule, which contains one or more epitopes. In many cases,antigens are also immunogens, thus the term “antigen” is often usedinterchangeably with the term “immunogen.” The substance may then beused as an antigen in an assay to detect the presence of appropriateantibodies in the serum of the immunized animal.

The term “antigenic determinant” as used herein refers to that portionof an antigen that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the “immunogen” used to elicitthe immune response) for binding to an antibody.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general.

As used herein, the term “adjuvant” is defined as a substance known toincrease the immune response to other antigens when administered withother antigens. If adjuvant is used, it is not intended that the presentinvention be limited to any particular type of adjuvant—or that the sameadjuvant, once used, be used all the time. It is contemplated thatadjuvants may be used either separately or in combination. The presentinvention contemplates all types of adjuvant, including but not limitedto agar beads, aluminum hydroxide or phosphate (alum), IncompleteFreund's adjuvant (incomplete or complete), as well as Quil A adjuvantand Gerbu adjuvant (Accurate Chemical and Scientific Corporation), andbacterins (i.e., killed preparations of bacterial cells). It is furthercontemplated that the vaccine comprise at least one “excipient” (i.e., apharmaceutically acceptable carrier or substance) suitable foradministration to a human or other animal subject. It is intended thatthe term “excipient” encompass liquids, as well as solids, and colloidalsuspensions.

As used herein the term “immunogenically-effective amount” refers tothat amount of an immunogen required to invoke the production ofprotective levels of antibodies in a host upon vaccination.

The term “protective level,” when used in reference to the level ofantibodies induced upon immunization of the host with an immunogen meansa level of circulating antibodies sufficient to protect the host fromchallenge with a lethal dose of an organism or other material (e.g.,toxins, etc.).

The terms “self antigen” or “autoantigen” refer to an antigen or amolecule normally expressed by an individual, but which solicits animmune response. Under normal conditions, these autoantigens arerecognized during an immune response as self (i.e., an antigen that isnormally part of the individual), and do not solicit an immune response.This is in contrast to antigens which are foreign, or exogenous, and arethus not normally part of the individual's antigenic makeup. “Selfantigen” or “autoantigen” is recognized as foreign, although the antigenis native to the individual's physiology.

As used herein, the term “autoimmune disease” means a set of sustainedorgan-specific or systemic clinical symptoms and signs associated withaltered immune homeostasis that is manifested by qualitative and/orquantitative defects of expressed autoimmune repertoires. Autoimmunediseases are characterized by antibody or cytotoxic immune responses toepitopes on self antigens. The immune system of the individual thenactivates an inflammatory cascade aimed at cells and tissues presentingthose specific self antigens. The destruction of the antigen, tissue,cell type, or organ attacked by the individual's own immune system givesrise to the signs and symptoms of the disease. Clinically significantautoimmune diseases include, for example, rheumatoid arthritis, multiplesclerosis, juvenile-onset diabetes, systemic lupus erythematosus (SLE),autoimmune uveoretinitis, autoimmune vasculitis, bullous pemphigus,myasthenia gravis, autoimmune thyroiditis or Hashimoto's disease,Sjogren's syndrome, granulomatous orchitis, autoimmune oophoritis,Crohn's disease, sarcoidosis, rheumatic carditis, ankylosingspondylitis, glomerulonephritis, Grave's disease, and autoimmunethrombocytopenic purpura.

The ability of a particular antigen to stimulate a cell-mediatedimmunological response may be determined by a number of assays,including but not limited to lymphoproliferation (ie., lymphocyteactivation) assays, CTL cytotoxic cell assays such as chromium-releaseassays, or by assaying for T lymphocytes specific for the antigen in asensitized subject. Such assays are well known in the art (See e.g.,Erickson et al., J. Immunol., 151:4189–4199 [1993] and Doe et al., Eur.J. Immunol., 24:2369–2376 [1994]).

The term “modulate,” as used herein, refers to a change in thebiological activity of a biologically active molecule. Modulation can bean increase or a decrease in activity, a change in bindingcharacteristics, or any other change in the biological, functional, orimmunological properties of biologically active molecules.

The term “agonist,” as used herein, refers to a molecule which, wheninteracting with an biologically active molecule, causes a change (e.g.,enhancement) in the biologically active molecule, which modulates theactivity of the biologically active molecule. Agonists may includeproteins, nucleic acids, carbohydrates, or any other molecules whichbind or interact with biologically active molecules. For example,agonists can alter the activity of gene transcription by interactingwith RNA polymerase directly or through a transcription factor.

The terms “antagonist” or “inhibitor,” as used herein, refer to amolecule which, when interacting with a biologically active molecule,blocks or modulates the biological activity of the biologically activemolecule. Antagonists and inhibitors may include proteins, nucleicacids, carbohydrates, or any other molecules that bind or interact withbiologically active molecules. Inhibitors and antagonists can effect thebiology of cells, tissues, organs or entire organisms.

The terms “Westem blot,” “Western immunoblot” “immunoblot” and “Western”refer to the immunological analysis of protein(s), polypeptides orpeptides that have been immobilized onto a membrane support. Theproteins are first resolved by polyacrylamide gel electrophoresis (i.e.,SDS-PAGE) to separate the proteins, followed by transfer of the proteinfrom the gel to a solid support, such as nitrocellulose or a nylonmembrane. The immobilized proteins are then exposed to an antibodyhaving reactivity towards an antigen of interest. In preferredembodiments, the binding of the antibody (i.e., the primary antibody) isdetected by use of a secondary antibody which specifically binds theprimary antibody. The secondary antibody is typically conjugated to anenzyme which permits visualization of the antigen-antibody complex bythe production of a colored reaction product or catalyzes a luminescentenzymatic reaction (e.g., the ECL reagent, Amersham).

As used herein, the term “ELISA” refers to enzyme-linked immunosorbentassay (or EIA). Numerous ELISA methods and applications are known in theart, and are described in many references (See e.g., Crowther,“Enzyme-Linked Immunosorbent Assay (ELISA),” in Molecular BiomethodsHandbook, Rapley et al. [eds.], pp. 595–617, Humana Press, Inc., Totowa,N.J. [1998]; See also, Harlow and Lane (eds.), Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory Press [1988]; and Ausubel et al.(eds.), Current Protocols in Molecular Biology, Ch. 11, John Wiley &Sons, Inc., New York [1994], for general descriptions of ELISAmethodology). In addition, there are numerous commercially availableELISA test systems, equipment and individual reagents.

One ELISA method is a “direct ELISA,” where an antigen (e.g., sTNFR1 orARTS-1) in a sample is detected. In one embodiment of the direct ELISA,a sample containing antigen is exposed to a solid (i.e., stationary orimmobilized) support (e.g., a microtiter plate well). The antigen withinthe sample becomes immobilized to the stationary phase, and is detecteddirectly using an enzyme-conjugated antibody specific for the antigen.

In an alternative embodiment, an antibody specific for an antigen isdetected in a sample. In this embodiment, a sample containing anantibody (e.g., anti-ARTS-1 antiserum) is immobilized to a solid support(e.g., a microtiter plate well). The antigen-specific antibody issubsequently detected using purified antigen and an enzyme-conjugatedantibody specific for the antigen.

In further alternative embodiments, an “indirect ELISA” is used todetect antibody or antigen in samples. In one embodiment, an antigen (orantibody) is immobilized to a solid support (e.g., a microtiter platewell) as in the direct ELISA, but is detected indirectly by first addingan antigen-specific antibody (or antigen), then followed by the additionof a detection antibody specific for the antibody that specificallybinds the antigen, also known as “species-specific” antibodies (e.g., agoat anti-rabbit antibody), which are available from variousmanufacturers known to those in the art (e.g., Santa Cruz Biotechnology;Zymed and Pharmingen/Transduction Laboratories).

In other embodiments, a “sandwich ELISA” is used, where the antigen isimmobilized on a solid support (e.g., a microtiter plate) via anantibody (i.e., a capture antibody) that is immobilized on the solidsupport and is able to bind the antigen of interest. Following theaffixing of a suitable capture antibody to the immobilized phase, asample is then added to the microtiter plate well, followed by washing.If the antigen of interest is present in the sample, it is bound to thecapture antibody present on the support. In some embodiments, a sandwichELISA is a “direct sandwich” ELISA, where the captured antigen isdetected directly by using an enzyme-conjugated antibody directedagainst the antigen. Alternatively, in other embodiments, a sandwichELISA is an “indirect sandwich” ELISA, where the captured antigen isdetected indirectly by using an antibody directed against the antigen,which is then detected by another enzyme-conjugated antibody which bindsthe antigen-specific antibody, thus forming anantibody-antigen-antibody-antibody complex. Suitable reporter reagentsare then added to detect the third antibody. Alternatively, in someembodiments, any number of additional antibodies are added as necessary,in order to detect the antigen-antibody complex. In some preferredembodiments, these additional antibodies are labelled or tagged, so asto permit their visualization and/or quantitation.

As used herein, the term “capture antibody” refers to an antibody thatis used in a sandwich ELISA to bind (i.e., capture) an antigen in asample prior to detection of the antigen. In one embodiment of thepresent invention, biotinylated capture antibodies are used in thepresent invention in conjunction with avidin-coated solid support.Another antibody (i.e., the detection antibody) is then used to bind anddetect the antigen-antibody complex, in effect forming a “sandwich”comprised of antibody-antigen-antibody (i.e., a sandwich ELISA).

As used herein, a “detection antibody” is an antibody which carries ameans for visualization or quantitation. Typically, detection antibodiesare conjugated enzyme moieties that yield a colored, fluorescent, orluminescent reaction product following the addition of a suitablesubstrate. Conjugated enzymes commonly used with detection antibodies inthe ELISA include, but are not limited to horseradish peroxidase,urease, alkaline phosphatase, glucoamylase and β-galactosidase. In someembodiments, the detection antibody is directed against the antigen ofinterest, while in other embodiments, the detection antibody is notdirected against the antigen of interest. Thus, in some embodiments, thedetection antibody is directed against another antibody. In someembodiments, the detection antibody is an anti-species antibody.Alternatively, the detection antibody is prepared with a label (e.g.,biotin, a fluorescent marker, or a radioisotope), and is detected and/orquantitated using this label.

As used herein, the terms “reporter reagent,” “reporter molecule,”“detection substrate” and “detection reagent” are used in reference toreagents which permit the detection and/or quantitation of an antibodybound to an antigen. For example, in some embodiments, the reporterreagent is a colorimetric substrate for an enzyme that has beenconjugated to an antibody. Addition of a suitable substrate to theantibody-enzyme conjugate results in the production of a colorimetric,flubrimetric, or luminescent signal (e.g., following the binding of theconjugated antibody to the antigen of interest). Other reporter reagentsinclude, but are not limited to, radioactive compounds. This definitionalso encompasses the use of biotin and avidin-based compounds (e.g.,including but not limited to neutravidin and streptavidin) as part ofthe detection system.

As used herein, the term “signal” is used generally in reference to anydetectable process that indicates that a reaction has occurred, forexample, binding of antibody to antigen. It is contemplated that signalsin the form of radioactivity, fluorimetric or colorimetricproducts/reagents will all find use with the present invention. Invarious embodiments of the present invention, the signal is assessedqualitatively, while in alternative embodiments, the signal is assessedquantitatively.

As used herein, the term “amplifier” is used in reference to a systemwhich enhances the signal in a detection method, such as an ELISA (e.g.,an alkaline phosphatase amplifier system used in an ELISA).

As used herein, the term “solid support” is used in reference to anysolid or stationary material to which reagents such as antibodies,antigens, and other test components are attached. For example, inpreferred ELISA methods, the wells of microtiter plates provide solidsupports. Other examples of solid supports include microscope slides,coverslips, beads, particles, cell culture flasks, as well as any othersuitable items.

As used herein, the term “kit” is used in reference to a combination ofreagents and other materials which facilitate sample analysis. Invarious embodiments of the present invention, a kit can includeantibodies (e.g., a suitable capture antibody, reporter antibody,primary antibody, secondary antibody, detection antibody) purifiedcontrol antigen, detection reagents, amplifier system, and nucleic acidprobes. Furthermore, in other embodiments, the kit includes, but is notlimited to, components such as apparatus for sample collection, sampletubes, holders, trays, racks, dishes, plates, instructions on the use ofthe kit, solutions or other chemical reagents, and samples to be usedfor standardization, normalization, and/or control samples. It iscontemplated that kits of the present invention can also includeapparatus and reagents for electrophoresis and blotting.

As used herein, the term “in vitro” refers to an artificial environmentand to processes or reactions that occur within an artificialenvironment. In vitro environments consist of, but are not limited to,controlled laboratory conditions. The term “in vivo” refers to thenatural environment (e.g., an animal or a cell) and to processes orreactions that occur within that natural environment.

As used herein, the terms “local” or “localized” and the like refer toconfinement to a small area, a single tissue, a single organ (e.g., alung) or other defined structure.

As used herein, the term “localized delivery” is delivery of an agent(e.g. a gene therapy agent or a drug) to a small area, a single tissue,a single organ or other specific structure. For example, localizeddelivery of a gene therapy agent to a single site (e.g., the liver) in asubject is typically achieved by injection into that site.

As used herein, the term “systemic” refers to multiple sites, tissues ororgans in an organism, or to the entire organism. Use of the word“systemic” generally indicates involvement of the circulatory orlymphatic systems.

As used herein, the term “systemic delivery” (in contrast to localizeddelivery) is delivery of an agent (e.g., a drug) to multiple sites,tissues or organs in an organism, or to the entire organism via thecirculatory system following an intravenous injection, or viagastrointestinal absorption of an orally administered agent.

As used herein, the term “surgical delivery” refers to the delivery ofan agent (e.g., a gene therapy agent) by surgical means (i.e., byoperation or some other invasive manipulation). Thus, in someembodiments, surgical techniques provide means for localized delivery ofan agent.

DESCRIPTION OF THE INVENTION

The present invention provides compositions and methods related toregulation of cytokine signaling through the Tumor Necrosis Factor (TNF)pathway. Specifically, the present invention provides a novelpolypeptide, and a gene which encodes the polypeptide, as set forth inFIG. 1 and SEQ ID NOS:1 and 2, which has the ability to promote theshedding of the extracellular domain of Type I Tumor Necrosis FactorReceptor (TNFR1). This polypeptide and gene are called ARTS-1, foraminopeptidase regulator of type I, 55 kDa tumor necrosis factorreceptor ectodomain shedding. It is contemplated that methods whichregulate the shedding of the sTNFR1 also regulate the activity of TNF.It is further contemplated that the ARTS-1 gene, as well as genessubstantially homologous to the ARTS-1 gene (and the gene product)regulate ectodomain shedding of other cytokine receptors, including IL-1and IL-6. It is also contemplated that the compositions and methodsprovided by the present invention will find use in therapeutics for thetreatment of diseases and disorders resulting from aberrant TNFactivity.

TNFR1 Shedding

The complete mechanisms underlying TNFR1 ectodomain shedding areunknown, but are thought to be mediated via proteolytic cleavage of thespacer region located between the transmembrane and TNF-ligand bindingdomain (Brakebusch et al., J. Biol. Chem., 269(51):32488–96 [1994]).Based upon amino acid sequencing of sTNFR1 isolated from urine, themajor cleavage site has been identified as occurring between Asn-180 andVal-181, with a minor site located between Lys-182 and Gly-183 (Nopharet al., EMBO J., 9:3269–3278 [1990]; Wallach et al., In Tumor NecrosisFactor: Structure-Function Relationship and Clinical Application (Osawaand Baonavida, eds.) Vol III, 47–57, S. Karger, Basel, Switzerland[1991]; and Brakebusch et al., J. Biol. Chem., 269(51):32488–96 [1994]).However, an understanding of the mechanism(s) is not necessary in orderto use the present invention.

Again, although an understanding of the mechanism(s) is not necessary inorder to use the present invention, a complete understanding of themechanism regulating TNFR1 shedding requires the identification andcharacterization of the interactions between regulatory proteins, suchas ARTS-1, TNFR1, and TNFR1 receptor sheddases, which appear to belongto the metalloprotease-disintegrin (ADAM) family of zincmetalloproteases (Blobel et al, Cell 90:589–592 [1997]). For example,TNF-α converting enzyme (TACE, ADAM17) has been reported to mediate theectodomain shedding of TNF-α, tranforming growth factor-α, L-selectinand TNFR2 (Black et al., Nature 385:729–733 [1997]; Moss et al., Nature385:733–736 [1997]; and Peschon et al., Science 282:1281–1284 [1998]).Similarly, TACE has been implicated in the regulated α-secretasecleavage of the amyloid precursor protein and ectodomain shedding oferbB4/HER4 (Buxbaum et al, J. Biol. Chem., 273:27765–27767 [1998]; Rioet al., J. Biol. Chem., 275:10379–10387 [2000]). Recent data suggestthat TACE may also mediate TNFR1 ectodomain shedding based upon thedemonstration of increased TNFR1 shedding in reconstitutedTACE-deficient cell lines (Reddy et al., J. Biol. Chem., 275:14608–14614[2000]). Indeed, TACE has been implicated as having sheddase activitytoward both TNFR1 and IL-1R type II (Reddy et al., supra).

Studies have also been undertaken to identify enzymes capable ofcleaving (i.e., shedding) the leucocyte selectin (L-selectin) receptor,a peripheral lymph node homing receptor. The enzymes which shed theL-selectin receptor have been theorized to be metalloproteases (Preeceet al., J. Biol. Chem., 271:11634–11640 [1996]; Peschon et al., Science282:1281–1284 [1998]; and Borland et al., J. Biol. Chem., 274:2810–2815[1999]).

Neither the sheddase nor the mechanism regulating the shedding of sTNFR1are known, although attempts have been made to identify sTNFR1 sheddingactivities in crude preparations. However, the results indicate metalrequirements for these activities, thereby tentatively categorizing theenzymes as metalloproteases (Bjonberg et al., Scand. J. Immunol.,42:418–424 [1995]; Mullberg et al., J. Immunol., 155:5198–5205 [1995];Katsura et al., Biochem. Biophys. Res. Comm., 222:298–302 [1996];Williams et al., J. Clin. Invest., 97(12):2833–2841 [1996]; andGallea-Robache et al., Cytokine 9:340–346 [1997]). However, themolecular identities of these metalloprotease enzymes remain unknown.

Björnberg et al. (Björnberg et al., Scand. J. Immunol., 42:418–424[1995]) indicate that in assays to identify and characterize enzymesinvolved in TNFR1 processing, inhibitors of aminopeptidases hadnegligible effects on release of sTNFR1. These authors indicate thataminopeptidases are not involved in release of sTNFR1.

The mechanisms which regulate TNF activity, TNFR activation, TNFR1signaling, sTNFR1 activity and sTNFR1 shedding are poorly understood.However, an understanding of these mechanisms is not necessary topractice the present invention. Indeed, the present invention is notlimited to any particular mechanism or mechanisms.

IL-1RII and IL-6R Shedding

As discussed above, the cognate receptors for the cytokines IL-1 andIL-6 also exhibit soluble forms akin to the soluble form of TNFR1.Furthermore, it has been suggested that these soluble receptor formsplay a role in the regulation of IL-1 and IL-6 activity andpro-inflammatory response. However, the proteins responsible for theshedding of ectodomains of the receptors for IL-1 and IL-6 remainunidentified. Nonetheless, it is contemplated that a protein whichregulates the shedding of TNFR1 ectodomain (e.g., a protein of thepresent invention) will also regulate the shedding of ectodomains ofother cytokine receptors, including IL-1RII and IL-6R. ARTS-1 binds tothe soluble form of the IL-6 receptor and promotes the shedding of theIL-6 receptor. ARTS-1 also binds to the soluble form of the type II IL-1receptors and promotes the shedding of the type II IL-1 receptors.

Anti-TNF Therapeutic Strategies

The recognition of TNF as an important inflammatory mediator in bothhealth and disease has fostered the development of a variety oftherapeutic strategies directed at inhibiting TNF bioactivity.

The benefits of inhibiting TNF activity during inflammatory reactionshave been demonstrated using neutralizing monoclonal antibodies to TNF(Tracey et al., Nature 330:662–664 [1987]; Hinshaw et al., Circ. Shock30:279–292 [1990]; Opal et al., J. Infect. Dis., 161:1148–1152 [1990];Silva et al., J. Infect. Sis., 162:421–427 [1990]; Emerson et al., Circ.Shock 38:75–84 [1992]; Fieldler et al., J. Lab. Clin. Med., 120:574–588[1992]; Jesmok et al., Am. J. Pathol., 141:1197–1207 [1992]; Walsh etal., Arch. Surg., 127:138–144 [1992]; and Williams et al., Proc. Natl.Acad. Sci. USA 89:9784–9788 [1992]). Unfortunately, the development ofwidespread clinical application of neutralizing monoclonal antibodiesdirected against human TNF is hampered by the potential for immunerejection of the mouse anti-TNF antibodies in human hosts. Thedevelopment of an immune response to non-human anti-TNF antibodiesadministered to human subjects may decrease the duration of therapeuticefficacy and also result in adverse events related to the formation ofimmune complexes or the development of hypersensitivity (Kempeni, Ann.Rheum. Dis., 58(S1):I73–I81 [1999]).

A chimeric monoclonal antibody consisting of the variable region of amurine anti-TNF monoclonal antibody fused to the constant region ofIgG1k has also been studied in clinical trials for the treatment ofCrohn's disease (i.e., inflammatory bowel disease) (Knight et al., Mol.Immunol., 30(16):1443–53 [1993]; Targan et al., N. Engl. J. Med.,337:1029–1035 [1997]; and U.S. Pat. No. 5,656,272 to Le et al., herebyincorporated by reference). However, this anti-TNF antibody is notoptimal, due to the likely development of human anti-chimeric antibodiesdirected against the non-human elements or the artificially fusedsequences within the chimeric anti-TNF antibody. These deleteriousantibodies are likely to reduce the half-life and therapeutic efficacyof the chimeric antibody, and possibly result in the formation ofunwanted immune complexes or the development of hypersensitivity (Targanet al., N. Engl. J. Med., 337:1029–1035 [1997]; Harriman et al., Ann.Rheum. Dis., 58:I61–I64 [1999]; and Kempeni, J. Ann. Rheum. Dis.,58:I73–I81 [1999]).

Anti-TNF therapies utilizing TNF receptor fragments and chimeric,soluble fusion proteins consisting of TNF receptor and IgG Fc, have beenreported to be efficacious for patients with rheumatoid arthritis andinflammatory bowel disease (Moreland et al., New Engl. J. Med.,337:141–147 [1997]; and U.S. Pat. No. 5,605,690 to Jacobs et al. (herebyincorporated by reference)). However, the therapeutic efficacy of theseanti-TNF therapeutic strategies utilizing soluble forms of the TNFreceptor or chimeric antibodies are also likely to be limited by thedevelopment of an immune response to the artificially fused humansequences (Kempeni, Ann. Rheum. Dis., 58(S1):I73–I81 [1999]).

Current methods of controlling TNF activity in patients also include theuse of non-specific inhibitors of cytokine gene transcription such ascorticosteroids (e.g., dexamethasone and cyclosporin-A). These methodshave significant and well known toxic side effects which result insignificant discomfort and potentially life-threatening susceptibilityto infection, as well as tissue and organ damage. Toxicities associatedwith cyclosporin-A include nephrotoxicity, hypertension, hepatictoxicity, neurotoxicity, hirsutism, gingival hyperplasia andgastrointestinal toxicity (Goodman & Gilman's The Pharmacological Basisof Therapeutics, 9^(th) edition, McGraw-Hill, NY, [1996], p. 1299).Toxicity associated with corticosteroids include adrenal suppression,hyperglycemia, hypertension, edema, hypokalemic alkalosis, myopathy,peptic ulcer disease, osteoporosis, aseptic (ischemic or avascular) bonenecrosis, mental status changes, glaucoma, cataracts and hyperlipidemia(Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9^(th)edition, McGraw-Hill, NY, 1996, p. 1475).

Induction of sTNFR1 shedding from cell culture systems can be induced bya variety of physiological and non-physiological mediators, such as,IFN-γ, IL-1β, IL-6, formyl-Met-Leu-Phe (fMLP), lipopolysaccharide,4b-phorbol 12-myristate 13-acetate (PMA), calcium ionophore,staurosporine, and sodium salicylate (Porteu et al., J. Biol. Chem.,266:18846–18853 [1991]; Nicod et al., Ann. NY Acad. Sci., 28:323–333[1994]; Tilg et al., Blood 83:113–118 [1994]; Zhang et al., J. Biol.Chem., 269:10270–10279 [1994]; Mullberg et al., J. Immunol.,155:5198–5205 [1995]; Levine et al., Am. J. Respir. Cell Mol. Biol.,14:254–261 [1996]; and Madge et al., J. Biol. Chem., 274:13643–13649[1999]). However, these agents are poor candidates for development asdrugs to regulate TNF activity in humans for a variety of reasons, mostnotably for the fact that most of these agents are toxic and notsuitable for human therapeutic use. In addition, most of these agentsare known to have or are likely to have detrimental or unwanted sideeffects resulting from their disruption of normal body processes inaddition to their effects on TNF signaling.

Other strategies for the regulation of TNF activity have also beenattempted or proposed. For example, U.S. Pat. Nos. 5,519,000 and5,641,751 to Heavner et al. (hereby incorporated by reference) describeshort (4–25 amino acid) peptides which bind TNF and inhibit TNFactivity. U.S. Pat. Nos. 5,665,859 and 5,766,917 to Wallach et al.(hereby incorporated by reference) describe methods to isolate a TNFR1protease or other polypeptides which influence TNFR1 shedding. U.S. Pat.No. 5,945,397 to Smith et al. (hereby incorporated by reference)describes human and mouse soluble TNF receptors and mutant variations ofthese receptors. U.S. Pat. No. 5,919,452 to Le et al. (herebyincorporated by reference) describes the use of anti-TNF antibodies,anti-TNF peptides, and soluble TNFR to treat TNF mediated pathologies.

However, until the development of the present invention, a nucleic acidin the form of a cDNA and a polypeptide encoded by the cDNA which hasthe ability to regulate the shedding of the TNFR1 ectodomain from theextracellular surface of a cell plasma membrane to yield a free, solubleform of the receptor, sTNFR1 had not been described. Indeed, databasesearches revealed that this gene and polypeptide had not heretofore beenidentified. These searches also revealed amino acid sequence motifsindicative of peptidase activity. Specifically, this gene has been namedaminopeptidase regulator of type I, 55 kDa tumor necrosis factorreceptor shedding, or “ARTS-1.” Methods for the use of the ARTS-1 gene,polypeptide and other related compositions are provided by the presentinvention. In addition, methods for the identification of genes andpolypeptides substantially homologous to ARTS-1 gene and polypeptide arealso provided by the present invention. However, an understanding of themechanisms of ARTS-1 activity is not necessary to practice the presentinvention.

It is contemplated that the protein (or proteins), and theircorresponding genes, which regulate the cleavage of TNFR1 from the cellsurface are likely to have important roles in regulating TNF activity invivo in health and disease states. It is contemplated that cleavage ofthe TNFR1 ectodomain from the cell surface limits TNF activity byproviding a pool of free receptors capable of binding and sequesteringTNF, as well as by removing functional TNFR1 from the cell surface. Itis further contemplated that occupation of the TNF bindings site on thefree sTNFR1 ectodomain does not activate the intracellular componentsinvolved in TNF signaling. It is further contemplated that the genes andproteins which regulate the shedding of soluble TNFR1 ectodomain willfind significant use in diagnostics and therapeutic regimens, as well asin the research setting. Furthermore, it is contemplated that genes andproteins which regulate the shedding of TNFR1 also regulate the sheddingof other cytokine receptors important in inflammatory diseases anddisorders (e.g., IL-1 and IL-6 receptors).

The present invention provides compositions and methods to identifygenes and proteins which regulate the cleavage and shedding of the TNFR1ectodomain. In addition, the present invention provides compositionssuitable for the production of monoclonal and/or polyclonal antibodiesdirected against the proteins involved in the shedding of cytokinereceptors (e.g., TNFR1). The present invention also providescompositions and methods suitable for the development of diagnostictools and assay systems for the assessment of the factors involved inregulation of soluble TNFR1 as an indicator of health and/or disease.

The present invention also provides compositions and methods suitablefor the development of more effective therapies for treating diseasesand disorders resulting from aberrant TNF activity (e.g., fordownregulation or upregulation of TNF activity), and consequentlyprovide therapeutic advantages for treatment of diseases or disordersresulting from inflammatory response or immune-deficiency.

In addition, the present invention provides compositions and methods todevelop additional means for suppressing damaging TNF-mediatedproinflammatory diseases or disorders without the toxic side effectsassociated with existing techniques for immune suppression. Thecompositions and methods provided by the present invention address theneed to identify genes and proteins which regulate TNF activity, andconsequently, have therapeutic value in the treatment of immunedisorders.

The remainder of the Description of the Invention is divided into thefollowing sections:

-   -   I) Cloning of Genes Encoding TNFR1Ectodomain Binding        Polypeptides    -   II) Preparation of Recombinant Vectors and Transformants    -   III) Analysis of ARTS-1 mRNA Expression    -   IV) Antibodies Directed Against ARTS-1 Polypeptide    -   V) Detection of ARTS-1 Polypeptide in Cultured and Primary Cells    -   VI) GST-ARTS-1 Polypeptide Expression and Purification    -   VII) Analysis of ARTS-1 Polypeptide Aminopeptidase Activity    -   VIII) Analysis of ARTS-1 TNFR1 Ectodomain Sheddase Regulatory        Activity    -   IX) Analysis of TNFR1 Ectodomain Sheddase Regulatory Activity of        ARTS-1 Catalytic Mutants    -   X) Demonstration of ARTS-1/TNFR1 Protein Interaction    -   XI) Therapeutic Agents for Immune Diseases and Disorders    -   XII) Diagnostic Agents for Immune Disease and Disorders    -   XIII) Identification of Genes Substantially Homologous to ARTS-1    -   XIV) Methods of Drug Screening

I. Cloning of Genes Encoding TNFR1 Ectodomain Binding Polypeptides

A) Yeast Two-Hybrid Screening

It was contemplated that polypeptides which form a physical interactionwith the ectodomain of TNFR1 would be potential candidates forpolypeptides which regulate the cleavage and shedding of the TNFR1ectodomain. A yeast two-hybrid screen was performed in order to identifysuch TNFR1 interacting polypeptides using a TNFR1 bait and human lungcDNA two-hybrid prey library. Yeast two-hybrid screening is a commontechnique in the molecular biology arts (Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, p. 20.0.1–20.1.40, John Wiley & Sons,Inc., New York [1994]). In the development of the present invention, theprotocols, yeast strains and reagents used in these experiments werethose supplied or recommended by the manufacturer (Matchmaker System 2;Yeastmaker Transformation System; Clontech), except where explicitlystated otherwise. The methods and compositions provided and used duringthe development of the present invention are provided in more detailbelow and in the Examples.

Conventional methods known to one of skill in the art may be used toprepare mRNA to construct a yeast-two hybrid library. In general, thesource of the mRNA (e.g., tissue or cultured cells) are treated with aguanidine reagent, phenol reagent or the like to obtain the total RNA.Subsequently, poly(A+)RNA (i.e., mRNA) is obtained therefrom by anaffinity column method using oligo dT-cellulose or poly U-Sepharosecarried on Sepharose 2B. Further, the resultant poly(A+) RNA can befurther fractionated by sucrose gradient centrifugation or the like.

Single-stranded cDNA is synthesized using the thus obtained mRNA as atemplate, an oligo(dT) primer and a reverse transcriptase. Then, adouble-stranded cDNA is synthesized from the resultant single-strandedcDNA. The resultant double-stranded cDNA is integrated (i.e., ligated)into an appropriate cloning vector. In the case of this yeast two-hybridlibrary, the resulting double-stranded cDNAs are cloned into the pGAD10plasmid (Clontech), a recombinant vector which contains operably linkedparts which enable the transcription and translation of a chimeric geneconsisting of the transcriptional activation domain of the yeast GAL4transcription factor and the subcloned cDNA. The pGAD10 vector containsadditional operably linked parts which enable its selection, replicationand propagation in yeast strains as well as E. coli.

As indicated above, the yeast two-hybrid screen was conducted using themanufacturer's recommended protocols. A bait vector was constructedusing the pAS2-1 plasmid (Clontech), to produce a chimeric fusion genecontaining nucleic acid sequences encoding the DNA binding domain ofGAL4 and the extracellular domain of the human TNFR1 receptor,corresponding to amino acids 26–216. Following sequential transformationof the chimeric pAS2-1 bait and pGAD10 prey vector library into yeaststrain Y190, the library was screened for clones positive for growth onhistidine (HIS) deficient media, followed by identification of thoseHIS⁺ clones which were also positive in a β-galactosidase filter liftassay. Thirty three clones positive for the ability to grow on HISdeficient substrate and β-galactosidase activity were identified, andeach of those clones was sequenced. It is noted that numerous protocolsand reagents for DNA sequencing are commercially available. It iscontemplated that any suitable method known in the art may readily beused in place of the protocol described here as well as other aspects ofthe production of the compositions of the present invention.

One positive clone, L26C-53A, was selected for further study. This clonecontained a 2355 bp insert containing a 631 amino acid open readingframe with a consensus zinc metalloprotease catalytic motif. Thenucleotide sequence of this clone corresponds to bases 1044 to 3082 ofthe nucleic acid of FIG. 1 (SEQ ID NO:1). This gene was namedaminopeptidase regulator of type I, 55 kDa tumor necrosis factorreceptor ectodomain shedding, or “ARTS-1.”

The yeast two-hybrid screening described herein is not meant to limitthe present invention to the particular reagents and methods described.Indeed, numerous other vectors and reagents may be used to produce theARTS-1 gene provided by the present invention. For example, a differenttwo-hybrid prey library may be substituted for the human lung cDNAlibrary to yield the ARTS-1 gene. Similarly, pAS2-1 and pGAD10 vectorvariants may be used, for example those which have different multiplecloning sites to facilitate subcloning of the cDNA insert. Vectorvariants which use the LexA operator components in place of the GAL4system may also be used in place of those described here. Also, variantsof these vectors with or without an operably joined HA antigenic tag maybe used in order to facilitate immunodetection of the fusion protein.Similarly, Saccharomyces cerevisiae strains other than Y190 may be usedas the doubly transformed host strain for the library screening (e.g.,S. cerevisiae strain CG-1945). In addition, selection conditions may bevaried, for example, by changing the concentration of 3-AT(3-amino-1,2,4-triazole) to counterselect leaky HIS⁺ clones. Many ofthese alternative variables and reagents are described and arecommercially available (e.g., from companies such as Stratagene andClontech). Thus, it is intended that the compositions of the presentinvention may be produced using any suitable method known in the art.

B) Phage Plaque Hybridization/cDNA Cloning

The DNA sequence identified in the yeast interaction screen appeared notto be a full length cDNA. This, the complete ARTS-1 cDNA was isolated byscreening a phage library using a ³²P-labelled DNA probe derived fromclone L26C-53A. The library used in this cDNA screening was constructedusing poly (A⁺) mRNA from the human NCI-H292 pulmonary mucoepidermoidcarcinoma cell line (ATCC, CRL 1848) which had been stimulated with 1 μMPMA (phorbol 12-myristate, 13-acetate, Sigma). This cell line has beendemonstrated to shed sTNFR1 in response to PMA stimulation (Levine etal., Am. J. Respir. Cell. Mol. Biol., 14:254–261 [1996]). The librarywas constructed using the uni-ZAP XR phage vector (Stratagene).

Bacteriophage from the library were plated in a lawn of XL1-Blue E. coli(Stratagene) at a density of 50,000 pfu per 150 mm plate and incubatedovernight at 37° C. Plaques were transferred to Hybond N+ filters(Amersham/Pharmacia) and denatured. Filters were then neutralized and UVcross-linked. Filters were washed in pre-hybridization solution, thenhybridized overnight at 42° C. with a ³²P-labelled L26C-53A insertgenerated by random primed labelling. Filters were washed, and were thenexposed to x-ray film overnight and positive plaques were selected.Positive plaques were subjected to two additional rounds of plaquehybridization prior to sequencing. Positive plaques were recovered viain vivo excision utilizing the ExAssist helper phage (Stratagene).

It is not intended that the present invention be limited to anyparticular cDNA library, reagent or method for the production of thenucleic acid encoding the ARTS-1 polypeptide of the present invention.Thus, it is contemplated that any suitable library will find use inisolating the ARTS-1 cDNA. For example, such libraries may includelibraries made from mRNA derived from human chronic myelogenous leukemiacell line K-562, lymphoblastic leukemia cell line MOLT-4 or lungcarcinoma A549 cell line. Similarly, multiple techniques for theradiolabelling and purification of nucleic acid probes as well as phageplaque hybridization protocols are well known in the art (Ausubel et al.(eds.), Current Protocols in Molecular Biology, Vol. 1–4, John Wiley &Sons, Inc., New York [1994]). The probe synthesis and hybridizationmethods and conditions described here are not meant to limit the scopeof the present invention. Variations on these protocols includealternative labelling methods (e.g., 5′ end labelling, overhang fill-inlabelling and random primed synthesis labelling). Alternative detectionsystems can also be used, including ³³P probe labelling andnon-radioactive probe visualization methods such as chemiluminescence.

Following three rounds of screening, four hybridizing phage clones wereidentified from the library, amplified, then sequenced. These fourclones all overlapped the L26C-53A sequence, as expected. However, noneencoded a full-length cDNA, nor did they collectively span the entiregene. One phage clone (bp 1–1777) contained the putative 5′ UTR andthree phage clones contained the putative 3′ UTR and the poly(A) tail(bp 2181–4845). cDNA sequence encoding the portion of the gene lyingbetween the 5′ and 3′ terminal clones was amplified via PCR from thesame human lung cDNA library using primers corresponding to 5′ and 3′sequence obtained from the phage screening. The cDNA segment amplifiedwith these primers was subcloned and both strands were sequenced toverify PCR fidelity.

As discussed in Section A above, it is not intended that the presentinvention be limited to any particular reagents, methods, and/orcompositions for the production of the nucleic acids and polypeptides ofthe present invention. Thus, it is contemplated that any suitablemethods for cDNA cloning and/or phage plaque hybridization will find usein the production of the compositions of the present invention. Forexample, one of numerous commercially available DNA polymerases may beused in the PCR reaction, including Taq (Stratagene, Promega), Pfu(Promega) or Sequenase (Amersham). Furthermore, it is contemplated thatalternative PCR reaction conditions (i.e., temperatures and timeintervals) will also be successful in amplifying the ARTS-1 nucleic acidof the present invention. Similarly, numerous vectors and reagents areequally suitable for subeloning and sequencing reactions.

C) Analysis of the ARTS-1 cDNA and Predicted Polypeptide

Inspection of the full length cDNA obtained as described above revealeda 4845 nucleotide transcript, containing a 2823 bp open reading frameand 5′ and 3′ untranslated regions as shown in FIG. 1. In this Figure, aconsensus polyadenylation site located at nucleotides 4795 to 4800 isindicated with a double underline and lies 18 nucleotides upstream of a27 nucleotide poly(A) tail. Two mRNA destabilization motifs are alsoidentified at nucleotides 3929 and 4457 using bold and underline.

The open reading frame (ORF) encodes a 941 amino acid polypeptide. Thefirst ATG codon lying in-frame relative to the largest open readingframe is located at nucleotide 88, and a TAA stop codon in the samereading frame is located at nucleotide 2911. Asparagine residuescomprising five potential N-glycosylation sites are indicated withcircles. A putative transmembrane domain, extending from amino acids 5to 28, is also indicated in the Figure with a single underline. In orderto determine this sequence, sequence analysis, including Kyte-Doolittlehydropathy prediction (Kyte and Doolittle, J. Mol. Biol., 157:105–132[1982]) was performed using MacVector 7.0 software (Oxford Molecular).The location of the putative hydrophobic transmembrane α-helical domainwas predicted utilizing several web-based analysis programs (MEMSAT2(McGuffin et al., Bioinform., 16:404–405 [2000]; Sosui (Hirokawa et al.,Bioinform., 14:378–379 [1998]; TMAP (Persson and Argos, J. Mol. Biol.,237:182–192 [1994]); TMpred (Hofmann and Stoffel, Biol. Chem.Hoppe-Seyler 374:166 [1993]; and TopPred2 (von Heijne, J. Mol. Biol.,225:487–494 [1992]). In sum, ARTS-1 is predicted to be a type IIintegral membrane protein with a single hydrophobic transmembraneα-helical domain, located between amino acids 5 and 28 (See, FIGS. 1 and2), and a very short hydrophobic intracellular amino-terminal domain(See, McGuffin et al., Bioinform., 16:404–405 [2000]; Hirokawa et al.,Bioinform., 14:378–379 [1998]; Persson and Argos, J. Mol. Biol.,237:182–192 [1994]); Hofinann and Stoffel, Biol. Chem. Hoppe-Seyler374:166 [1993]; and von Heijne, J. Mol. Biol., 225:487–494 [1992]).

Subdomains of homology indicate that ARTS-1 is a member of theaminopeptidase family of gluzincin zinc metalloproteases. This family ofproteins share motifs indicating similar biochemical activity, althoughthe family members are extremely diverse in overall structure inbiological function (Zinc Metalloproteases in Health and Disease, Taylor& Francis, London, England [1996], Hooper, p. 1–21, and Wang and Cooper,p. 131–151). FIG. 1 also indicates the ARTS-1 sequences which adhere tothe consensus zinc metalloprotease catalytic motif for theaminopeptidase family, HEXXH(Y)₁₈E (SEQ ID NO:10). Within this motif, itis theorized that the two histidine residues (H) and the second glutamicacid residue (E) represent the zinc binding domain while the firstglutamic acid mediates the catalytic activity. In the ARTS-1polypeptide, this consensus motif is observed at T³⁵⁰VAHELAHQWFG (SEQ IDNO:8) and L³⁷²WLNEGFA (SEQ ID NO:9) (boxed in FIG. 1).

Furthermore, a schematic representation of the ARTS-1 protein,indicating domains of homology with the aminopeptidase family ofgluzincin zinc metalloproteases is provided in FIG. 2. A zincmetalloprotease consensus catalytic motif HEXXH(Y)₁₈E (SEQ ID NO:10), ashort intracytoplasmic tail, a transmembrane domain and a large 375amino acid domain of homology are also depicted in this Figure.

Quantitative comparisons between ARTS-1 protein and other members of theaminopeptidase-gluzincin zinc metalloprotease family are shown in Tables1 and 2 below. Table 1 provides quantitation of percent identity andpercent similarity between the full length amino acid sequence of theARTS-1 protein and other aminopeptidase family members. Percentidentities are shown above the shaded diagonal and percent similaritiesare shown below the shaded diagonal. Table 2 shows a similar comparisonbetween the conserved 375 amino acid domain in the ARTS-1 proteincontaining the consensus zinc binding motif HEXXH(Y)₁₈E (SEQ ID NO:10)and other members of the aminopeptidase family of gluzincin zincmetalloproteases. The aminopeptidase family members included in theseTables are human placental leucine aminopeptidase (PLAP) (Rogi et al.,J. Biol. Chem., 271:56–61 [1996]), rat insulin-regulated aminopeptidase(IRAP) (Keller et al., J. Biol. Chem., 270:23612–23618 [1995]), humanaminopeptidase A (AMP A) (Nanus et al., Proc. Natl. Acad. Sci. USA90:7069–7073 [1993]; and Li et al., Genomics 17:657–664 [1993]), humanaminopeptidase N (AMP N) (Olsen et al., FEBS Lett., 238:307–314 [1988]),human puromycin sensitive aminopeptidase (Tobler et al., J. Neurochem.,68:889–897 [1997]), rat thyrotropin-releasing hormone degrading enzyme(TRH DE) (Schauder et al., Proc. Natl. Acad. Sci. USA 91:9534–9538[1994]), S. cerevisiae aminopeptidase YSCII (Garcia-Alvarez et al, Eur.J. Biochem., 202:993–1002 [1991]), C. elegans cosmid F49E8.3 geneproduct (Wilson et al., Nature 368:32–38 [1994]), and Lactococcus lactisaminopeptidase N (Tan et al., FEBS Lett., 306:9–16 [1992]).

As mentioned above, and as indicated in these Tables, although membersof this aminopeptidase family share a similar biochemical motif, thereis significant sequence divergence among family members, likelyindicating distinct and specialized biological functions.

TABLE 1 Percentage Identity and Similarity Between Full Length ARTS-1Protein and Other Members of the Aminopeptidase-Gluzincin ZincMetalloprotease Family Human Human Rat Human Human Rat Human C. elegansL. Lactis S. cerevisiae Protein ARTS-1 PLAP IRAP AMP A AMP N TRH DE PSAF49E8.3 AMP N YSC II Human 44 41 31 29 27 30 26 24 27 ARTS-1 Human 17 7930 29 27 28 25 23 26 PLAP Rat 15 5 28 28 28 26 24 21 24 IRAP Human 20 1918 34 29 30 25 22 28 AMP A Human 18 18 18 18 22 29 27 24 26 AMP N Rat 1716 17 17 14 26 23 22 22 TRH DE Human 17 19 17 16 17 16 36 28 32 PSA C.elegans 18 17 15 18 17 17 16 28 31 F49E8.3 L. lactis 16 16 15 17 15 1416 18 27 AMP N S. cerevisiae 16 18 16 15 16 16 16 19 18 YSC II

TABLE 2 Percentage Identity and Similarity Between the Conserved DomainsContaining the Zinc Binding Motif of ARTS-1 Protein and Other Members ofthe Aminopeptidase-Gluzincin Zinc Metalloprotease Family Amino AcidHuman Human Rat Human Human Rat Human C. elegans L. lactis S. cerevisiaeProtein Position ARTS-1 PLAP IRAP AMP A AMP N TRH DE PSA F49E8.3 AMP NYSC II Human 161–535 51 53 39 43 41 44 42 36 41 ARTS-1 Human 192–540 1590 42 43 39 44 44 36 44 PLAP Rat 273–621 12 6 41 42 39 44 45 37 43 IRAPHuman 202–549 21 18 18 44 41 48 44 36 45 AMF A Human 192–554 15 15 16 1942 47 44 37 45 AMP N Rat 248–603 14 17 17 17 16 41 40 37 38 TRH DE Human158–508 16 18 17 14 14 18 52 41 52 PSA C. elgens 120–469 16 17 16 17 1720 16 41 49 F49E8.3 L. Lactis  99–445 16 20 18 18 17 15 18 17 40 AMP NS. cerevisiae 114–461 15 16 15 16 16 19 15 18 20 YSC II

II. Preparation of Recombinant Vectors and Transformants

Preparation of Recombinant Vectors

Numerous recombinant vectors relevant to the present invention arecontemplated. Such vectors, containing the gene or portions of the geneof the present invention (i.e., the ARTS-1 gene) may be of any type,with the only limitation being that the vector into which the gene ofthe invention has been inserted has the capacity for replication in ahost. As used in the present invention, a host may be a bacteria (e.g.,E. coli BL21 strain), a yeast (e.g., S. cerevisiae Y190 strain), or ananimal cell (e.g., the NCI-H292 human pulmonary mucoepidermoid carcinomacell line). Furthermore, the host cell may exist in an in vitro tissueculture system, or exist within an intact organism (e.g., a mouse or ahuman). Indeed, it is not intended that the host be limited to anyparticular cell type, nor is it intended that the cell host bemaintained in any particular setting.

Protocols for the construction of recombinant vectors are commonplace inthe molecular biology arts, and reagents for the manipulation ofrecombinant nucleic acids are readily available from commercial sources.Routine procedures in the construction of a recombinant vectorencompassed by the present invention may include restrictionendonuclease digestion, ligation, phosphorylation, dephosphorylation,blunt-ending, size separation, annealing, eluting, staining, desalting,transformation, inoculating, incubating, cesium banding and purificationby a variety of commercially available products. In the most simple ofembodiments, a recombinant vector of the invention may be made bydigesting the gene of the invention with an appropriate restrictionenzyme, ligating into an appropriately digested vector, transforming theligated vector into bacteria, selection using an antibiotic resistancemarker contained on the vector and purifying the vector using a kitdesigned for rapid, small scale plasmid purification.

A vector may be in the form of a DNA plasmid. Such plasmids containmultiple parts operably arranged to permit replication in a minimum ofone specific host species. A plasmid (e.g., the pGEX-6P-1 plasmid) maybe restricted to replication in bacteria, or may also contain operablylinked sequences which permit the plasmid to propagate or function in asecond species in addition to its usual bacterial host. For example, thepAS2-1 and pGAD10 vectors contain operably linked nucleic acid sequenceswhich permit their propagation in both bacteria and yeast (e.g., the S.cerevisiae Y190 strain), while the pTarget vector contains operablylinked sequences which permit its propagation in a bacterial host, butalso contains sequences which allow it to direct the transcription of acloned gene in a mammalian cell.

A vector may also be in the form of a DNA or RNA bacteriophage or othervirus, which further may be in the form of a DNA or RNA containingvirus. Such phage or other virus may be replication competent orreplication defective. The phage or viral nucleic acid may also beengineered to permit propagation in an organism as a plasmid in additionto its viral life cycle. For example, the bacteriophage Lambda (λ)uni-ZAP II vector (Stratagene) is capable of bacterial infection as abacteriophage, but also contains sequences which enable the excision andpropagation of a plasmid form of the vector. Such a vector is oftencalled a “phagemid.” Further, recombinant viral vectors (e.g.,retrovirus, adenovirus, adeno-associated virus or vaccinia virus) or aninsect virus vector (e.g., baculovirus) may also be used to infectcells.

In particularly preferred embodiments, the gene of the present inventionis operably linked to other components of the vector. For this purpose,the vector of the invention may contain, if desired, cis elements (e.g.,an enhancer, splicing signal, poly(A) addition signal, selection marker,ribosome binding sequence (i.e., Shine-Dalgarno or Kozak sequences) orthe like) in addition to a promoter/enhancer and the gene of the presentinvention. As the selection marker, genes encoding resistance todihydrofolate reductase, ampicillin, kanamycin, neomycin, or the likemay be used.

Preparation of Transformants

In some embodiments of the present invention, a transformant wasobtained by introducing the recombinant vector containing the gene ofthe invention into a host. The host is not particularly limited as longas it can harbor the vector of the invention. Specific examples of hostinclude Escherichia bacteria such as E. coli; yeast such as S.cerevisiae and Schizosaccharomyces pombe; animal cells such as COScells, CHO cells, NCI-H292 cells or non-specified cells of an intactorganism; or insect cells such as Sƒ9 and Sƒ21 cells. In someembodiments of the present invention, the vector is introduced into thehost under conditions such that the polypeptide encoded by the gene ofthe invention is expressed.

In some cases, when a bacterium such as E. coli is used as the host, therecombinant vector of the invention is capable of replication in thehost and, at the same time, is capable of expressing the gene of theinvention within the bacterial host (e.g. the pGEX-ARTS-1 vectors). Suchvectors in general preferably consist of a promoter, a ribosome bindingsequence, the gene of the present invention and a transcriptiontermination sequence. The vector may also contain a gene to control thepromoter.

Any promoter may be used as long as it can appropriately direct theexpression of the gene of interest in the host cells, such as amammalian cell or E. coli. For example, an E. coli or phage-derivedpromoter such as trp promoter, lac promoter, P_(L) promoter or P_(R)promoter may be used. An artificially designed and altered promoter suchas tac promoter may also be used.

Any method for bacterial transformation may be used for introducing arecombinant vector into a bacterium. Many methods are commonly known inthe art, including electroporation and the use of bacteria madecompetent by calcium chloride (See e.g., Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, p. 1.8.1–1.8.10, John Wiley & Sons,Inc., New York [1994]).

Likewise, any method for introducing DNA into yeast may be used tointroduce a recombinant vector into a yeast. For example,electroporation (Becker et al., Methods Enzymol., 194:182–187 [1991]),the spheroplast method (Hinnen et al., Proc. Natl. Acad. Sci. USA75:1929–1933 [1978]), the lithium acetate method (Ito, J. Bacteriol.,153:163–168 [1983]) or the like may be used.

It is contemplated that any suitable animal cells will find use as hostsin the present invention (e.g., simian COS-7 or Vero cells, Chinesehamster ovary cells (CHO cells), mouse L cells, rat GH3 cells, human FLcells, human lymphoid cell lines, or the like). A promoter/enhancer maybe used to express the ARTS-1 gene where the promoter is active in allor most cell types (e.g., the SRα promoter, SV40 promoter, HSV-LTRpromoter, CMV promoter or the like). Alternatively, promoter/enhancerelements may be used which direct expression of the ARTS-1 gene in onlya subset of cells (e.g., cells of the lymphoid system).

As with other host cells, any suitable method for introducing therecombinant vector into animal or insect cells (e.g., electroporation,the calcium phosphate method, lipofection or the like) may be used.

III. Analysis of ARTS-1 mRNA Expression

Following the identification and isolation of the ARTS-1 gene, anexperiment was undertaken to determine the pattern of tissue expressionof the endogenous ARTS-1 mRNA. This was accomplished using amulti-tissue Northern blot and an ARTS-1 derived probe as shown in FIG.3 and described in greater detail in Example 3.

The blot used in this experiment was a commercially available humanmultiple tissue poly(A+) Northern blot (Clontech). This blot was probedaccording to the manufacturer's suggested protocol using a full length³²P-labelled ARTS-1 cDNA probe.

The Northern blot analysis shown in FIG. 3, Panel A, indicates that thehuman ARTS-1 transcript was expressed in multiple tissues, includingspleen, thymus, small and large intestine, peripheral blood leukocyte,heart, placenta, lung, skeletal muscle, kidney and pancreas. In thesetissues, a single predominant mRNA species of approximately 5.7 kB wasdetected. FIG. 3, Panel B, shows the same blot following stripping andrehybridization to a probe specific for the human GAPDH transcript as areference for RNA loading normalization.

Furthermore, in view of numerous alternative protocols known in the artfor Northern blotting, it is not intended that the present invention belimited to the Northern blotting protocol provided in Example 3 or anyother particular Northern blotting method. For example, in someembodiments, RNA is isolated from tissue samples using alternativemethods (e.g., a commercial RNA isolation kit such as Qiagen RNeasyTotal RNA Mini Kit, Catalog No. 74103).

Similarly, alternative probe synthesis and labelling techniques alsofind use with the present invention. For example, any probe having aminimum complementarity of 25 base pairs to the ARTS-1 cDNA will finduse in the Northern blot methods of the present invention. Furthermore,it is contemplated that the nucleic acid comprising the probe will begenerated by PCR, by restriction digest, or by synthetic oligonucleotidesynthesis. Alternative nucleic acid probe labelling methods also finduse with the present invention (e g., labelling with ³³P radioisotope ornon-radioactive labelling methods). In addition, alternative Northernblotting protocols, reagents and equipment suitable for use in thepresent invention are known in the art (See, e.g., Ausubel et al.(eds.), Current Protocols in Molecular Biology, Vol. 1, pages4.9.1–4.9.16, John Wiley & Sons, Inc., New York [1994]).

IV. Antibodies Directed against the ARTS-1 Polypeptide

In the present invention, polyclonal antiserum directed against theARTS-1 polypeptide is provided. However, the antibody of the inventionmay be prepared by various methods, as numerous methods for theproduction of monoclonal and polyclonal antibodies are well known in theart (See e.g., Sambrook, J. et al. (eds.), Molecular Cloning, ColdSpring Harbor Laboratory Press [1989]; Harlow and Lane (eds.),Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press[1988]; Ausubel et al. (eds.), Current Protocols in Molecular Biology,p. 11.4.2–11.15.4, John Wiley & Sons, Inc., New York [1994]). As usedherein, the term “antibody” means an antibody molecule as a whole or afragment of an antibody (e.g., Fab or F(ab′)₂ fragment) which can bindspecifically to an antigen. The antibody provided by the invention isdescribed in more detail below and in Example 4. The present inventionalso contemplates that these same methods will find use in theproduction of antibodies specific for polypeptides encoded by geneswhich are substantially homologous to the ARTS-1 gene.

In order to conduct additional studies on the ARTS-1 polypeptide,polyclonal antiserum was generated against an ARTS-1 polypeptide. Acommercial service (Research Genetics) was used in these studies.Specifically, a 17 amino acid ARTS-1 synthetic peptide was used toimmunize New Zealand white rabbits. This peptide corresponded to aminoacids 538 to 554 of the ARTS-1 polypeptide and had the sequence:

-   -   R⁵³⁸GRNVHMKQEHYMKGSD (SEQ ID NO:7)        This particular peptide was chosen based upon its antigenic        potential and its lack of homology with other protein sequences        as determined by a BLAST homology search.

Rabbits were immunized with this peptide using standard techniques. TheARTS-1 peptide was conjugated to KLH and mixed with an equal volume ofFreund's complete adjuvant. The amount of antigen utilized perimmunization was 0.1 mg, which was injected into three subcutaneousdorsal sites. The animals received boosts at weeks 2, 6 and 8. Bleedswere obtained at weeks 4, 8 and 10, and tested for the presence ofanti-ARTS-1 antibody. In subsequent experiments, the antiserum obtainedfrom the 10 week bleed was used.

In view of numerous alternative protocols known in the art for theproduction of polyclonal antibodies, the present invention is not meantto be limited to any particular method. For example, the entire 941amino acid ARTS-1 polypeptide, or any portion or fragment thereof, maypotentially be used as the immunogen, and the immunogen may be eithersynthetic or native. It is not intended that the present invention belimited to any particular ARTS-1 derived immunogen, method ofimmunization, immunization schedule, animal species, test protocol fordetermining antibody production or antibody purification method.

Although the antibody provided by the invention is polyclonal, theinvention also contemplates monoclonal antibodies directed against theARTS-1 polypeptide. It is also contemplated that any suitable method forthe production of monoclonal antibodies will find equal use in thepresent invention. In one embodiment, the immunogen used to producethese monoclonal antibodies comprises full-length ARTS-1 polypeptide,although it is contemplated that any portion or fragment of the ARTS-1polypeptide may also find use in the present invention as an imnmunogen.

In these monoclonal antibody protocols, any suitable method for recoveryof antibody producing cells, cell fusion, selection and cloning ofhybridomas, recovery of the monoclonal antibodies, and purification ofthe monoclonal antibodies of interest may be used. Thus, it is notintended that the present invention be limited to any particularmonoclonal antibody production system or method.

If desired, the polyclonal or monoclonal antibody preparation of theinvention can be purified from crude antiserum or culture supernatantusing a conventional method (e.g., Protein A affinity, ammonium sulfateprecipitation, ion exchange chromatography, gel filtration, affinitychromatography, or any of these methods in combination).

Once the polyclonal or monoclonal antibody is thus obtained, in someembodiments of the present invention, the antigen is bound to a solidsupport, so as to thereby prepare an affinity chromatography column.Using this column, the antibody of the present invention can be highlypurified. Conversely, in an alternative embodiment of the presentinvention, the antibody is bound to a solid support as to therebyprepare an affinity chromatography column. Using this column, the ARTS-1polypeptide, or fragments thereof, either native or recombinant, can behighly purified from variable sources. These monoclonal and polyclonalantibodies find numerous uses, including Western blotting,immunoprecipitation, immunohistochemistry and clinical applicationsusing methods known in the art (See e.g., Harlow and Lane (eds.),Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press[1988]; Ausubel et al. (eds.), Current Protocols in Molecular Biology,Vol. 1–4, John Wiley & Sons, Inc., New York [1994]; and Laurino et al.,Ann. Clin. Lab Sci., 29(3):158–166 [19991]).

V. Detection of ARTS-1 Polypeptide in Cultured Cell Lines and PrimaryCells

Following the production of ARTS-1 polyclonal antiserum, the expressionof endogenous ARTS-1 polypeptide in cultured and primary cells wasinvestigated using standard Western immunoblotting techniques well knownin the art, as discussed below and described in further detail inExample 5. Protein concentrations of the samples were assayed and 20micrograms of total protein were prepared for analysis in Laemmlibuffer. Samples were resolved via 6% polyacrylamide SDS-PAGE andelectroblotted onto nitrocellulose. Blots were incubated overnight inblocking buffer, then incubated for 2 hours with ARTS-1 antiserum at a1:20,000 dilution in blocking buffer. Membranes were washed, thenincubated with horseradish peroxidase conjugated goat anti-rabbit IgG(Life Technologies) diluted to 1:5,000 in blocking buffer, then washedagain. Membranes were then incubated in chemiluminescent detectionsubstrate and the signal detected on X-ray film.

The specificity of the resulting polyclonal antiserum was firstdetermined as shown in FIG. 4. Extracts analyzed in this experiment werecrude whole cell homogenates, and membrane and cytosolic fractions allprepared from cultured NCI-H292 cells. As shown in FIG. 4, Panel A, theanti-ARTS-1 antiserum detected a predominant 100 kDa membrane form and apredominant 68 kDa cytosolic form from the NCI-H292 cells, while thewhole cell extracts predictably revealed a mixture of these two forms.As shown in the second panel FIG. 4, the preimmune serum showed noreactivity towards the same samples when used at the same concentration.

Specificity of the immune serum was further demonstrated in competitionexperiments in the third and fourth panels of FIG. 4. Preincubation ofthe immune serum with the RGRNVHMKQEHYMKGSD peptide (SEQ ID NO:7)against which the polyclonal antiserum was raised resulted in almostcomplete attenuation of the immune signal (Bottom panel). In contrast,preincubation of the immune serum with bovine serum albumin resulted inminimal attenuation of immune signal (Third panel).

The expression of endogenous ARTS-1 polypeptide in primary cells andother cell lines was further investigated using the identical antiserumand Western immunoblot technique (See, FIG. 5). These experiments wereconducted to determine if there were differences in the ARTS-1polypeptide forms expressed in different cell lines. These experimentsanalyzed membrane and cytosolic fractions made from human bronchialbrushing specimens, airway epithelial cell lines BEAS-2B and BET-1A,human lung carcinoma cell line A549, cultured NCI-H292 cells, primarycultures of normal human bronchial epithelial cells (NHBE), humanumbilical vein endothelial cells (HUVEC) and human fibroblasts.

Similar to the NCI-H292 cell line, the BEAS-2B, BET-1A, NHBE and A549cell lines all revealed a 100 kDa band localized predominantly in themembrane fraction, and a 68 kDa form expressed to varying degrees in thecytosolic fraction. The protein samples obtained from human bronchialbrushings, as well as the HUVEC and fibroblast primary cells revealedsimilar patterns, with the exception that both the 100 and 68 kDa formsappear in the membrane fraction. The human bronchial brush cells andoccasionally the NCI-H292 cell line also showed a larger 132 kDa form.

These multiple sized forms may be due to regulated processing of theARTS-1 polypeptide. Furthermore, the distinction between the differentsized ARTS-1 forms seen in the membrane versus the cytosolic fractionsmay also indicate regulated processing between the membrane and cytosol.However, it is not necessary to understand the mechanism of ARTS-1processing or localization in order to practice the present invention,nor is it intended that the present invention be so limited.

Furthermore, in view of numerous alternative protocols known in the artfor Western blotting, it is not intended that the present invention belimited to the Western blotting protocol provided in Example 5 or anyother Western blotting method. For example, in some embodiments,alternative secondary (i.e., detection) antibodies can be used.Alternative Western irmnunoblotting protocols, reagents and equipmentsuitable for use in the present invention are known in the art (See,e.g., Ausubel et al. (eds.), Current Protocols in Molecular Biology,Section 10.8, “Immunoblotting and Inmmunodetection,” John Wiley & Sons,Inc., New York [1994]).

VI. GST-ARTS-1 Polypeptide Expression and Purification

In order to facilitate the analysis of ARTS-1 biochemical activity, ahighly purified form of the ARTS-1 polypeptide was produced using a GSTfusion protein protocol commonly used in the art. The specifics of thisprotocol are provided in detail in Exarnple 6. It is not intended thatthe present invention be limited to any particular method for GST-ARTS-1polypeptide expression, either with or without subsequent GST-ARTS-1polypeptide purification.

The use of GST fusion proteins to produce purified polypeptides iscommon in the art. In these protocols, a transcriptional andtranslational fusion is made between the genes encodingglutathione-S-transferase (GST) and the gene of interest (e.g. theARTS-1 gene). Production of the fusion protein containing a GST “tag”enables the effective and rapid purification of the fusion protein byuse of an affinity column comprising immobilized glutathione. In orderto produce a GST-ARTS-1 polypeptide, the cDNA sequence encoding ARTS-1was first PCR amplified and subeloned into the pGEX plasmid backbone(Amersham Pharmacia) and used to transform the BL21 E. coli host strain.The pGEX plasmid contains a multiple cloning site and expresses a fusionprotein consisting of the GST polypeptide and a subdloned codingsequence (e.g., the ARTS-1 coding sequence) cloned into the multiplecloning site of the plasmid. Transcription of the fusion gene iscontrolled by the conditional tac promoter. In accordance with themanufacturer's protocol, clones were cultured in the presence of 0.6 mMisopropyl β-D-thiogalactoside (IPTG) and subsequently lysed with aprotein extraction buffer. Transformed clones expressing GST-ARTS-1fusion protein were selected by Western immunoblotting utilizing ananti-GST antibody (Amersham Pharmacia). Cells with confirmed expressionof GST-ARTS-1 polypeptide were lysed and centrifuged to separate thesoluble from the insoluble fractions. The GST-ARTS-1 fusion protein wasisolated from the insoluble fraction by denaturation with 6M urea, thendesalted and renatured. The GST-ARTS-1 fusion protein was purifiedutilizing a glutathione sepharose 4B affinity column using techniqueswell known in the art.

To assess the purity of the eluted recombinant GST-ARTS-1 fusionprotein, samples were subjected to SDS-PAGE and stained with Coomassiebrilliant blue, as shown in FIG. 6A. In this Figure, soluble andinsoluble protein fractions from BL21 E. coli transformed with emptypGEX vector are shown in lanes 1–2, and analogous samples from bacteriacontaining the pGEX-ARTS-1 vector are shown in lanes 3–4. The GST-ARTS-1fusion protein following elution is shown in lane 5, as a predominant132 kDa band, corresponding to the predicted molecular weight of aGST-ARTS-1 fusion protein (104 kDa ARTS-1 extracellular domain plus 26kDa GST tag). The control purified GST tag was revealed as a predicted26 kDa band in lane 6.

The recombinant purified GST-ARTS-1 fusion protein samples were furthersubjected to FPLC analysis to assess their purity, the results of whichare shown in FIG. 6B. Elution from the FPLC column was monitored byabsorbance at a wavelength of 280 nm. This analysis of the purifiedGST-ARTS-1 revealed a single major protein elution peak at approximately40 minutes.

Using the aminopeptidase activity assay described below, FPLC fractionswere assessed for aminopeptidase activity utilizing a phenylalaninep-nitroaniline substrate. The results of these experiments are shown inFIG. 6C. As indicated in this Figure, the single major protein peakrevealed by FPLC analysis coeluted with a peak of aminopeptidaseactivity against a phenylalanine-pNA substrate in pooled fractions from38–44 minutes.

As those of skill in the art know, numerous protocols for thepurification of polypeptides are available. The use of numerousalternative protocols for the production of isolated ARTS-1 polypeptideare easily envisioned. These alternative protocols may be used to purifyARTS-1 polypeptides other than the GST-ARTS-1 polypeptide described bythe present example. Indeed, it is not intended that the presentinvention be limited to any particular protocol.

Numerous reagents and variables may be manipulated or substituted forthose used in Example 6 to yield a substantially similar purified ARTS-1polypeptide. Such variables include the choice of ARTS-1 polypeptide(e.g., either with or without a fusion protein tag), the cells used forthe production of the starting materials, the promoters/enhancers usedto drive expression of the recombinant protein to be purified, thecellular culture and growth conditions, harvesting methods, mode ofpurification, and methods to assess polypeptide purity followingpurification. The detailed protocol provided in Example 6 is not meantto limit the scope of the present invention. Indeed, it is contemplatedthat any protocol which will produce a substantially similar purifiedproduct will find use with the present invention. Such alternativeembodiments are briefly discussed below.

Alternative protocols may be used to purify forms of ARTS-1 polypeptideother than the GST-ARTS-1 fusion polypeptide provided by the presentinvention. These alternative forms may include a maltose binding protein(MBP) ARTS-1 fusion polypeptide, a polyhistidine (i.e., 6×His) taggedARTS-1 fusion polypeptide, a thioredoxin tagged ARTS-1 fusionpolypeptide, or a full length ARTS-1 polypeptide without any fused tagto facilitate purification.

Alternative protocols may find use in the present invention. Forexample, protocols which use different host systems as a source forstarting material (i.e., a source culture) for ARTS-1 purification maybe used. Such alternative source systems include insect cells with abaculovirus overexpression system (e.g., Sf9 or Sƒ21 cell lines),mammalian cell lines in conjunction with vectors designed forrecombinant polypeptide overexpression (expression vectors), ormammalian cells or tissues for the purification of ARTS-1 polypeptideexpressed from its endogenous (i.e., native) chromosomal location. Thecultivation of the transformed, transfected or infected host of theinvention is carried out in a medium and under conditions mostappropriate for the growth of that particular host cell. These mediaformulations and culture conditions are well known to those in the art.For example, culture of a mammalian cell line for the isolation of anoverexpressed or endogenous polypeptide will commonly use RPMI 1640 orDMEM media, typically supplemented with 5–10% fetal or newborn calfserum, and may be further supplemented with an antibiotic such askanamycin or penicillin. In some protocols, calf serum may be omitted tofacilitate subsequent polypeptide purification. Typically, thecultivation of mammalian cells is carried out in the presence 5% CO₂ at37° C.

Following the cultivation of a particular ARTS-1 polypeptide-containinghost, the proteins of the culture can be extracted by disrupting (by anysuitable method) the microorganisms or cells if the protein is producedwithin the host. If ARTS-1 polypeptide of the invention is secreted bythe host cells, the culture fluid is collected. The resultant cultureextract or culture supernatant is then subjected to conventionalbiochemical techniques used for protein purification. As known in theart, numerous techniques for polypeptide purification exist (e.g.,ammonium sulfate precipitation, gel chromatography, ion exchangechromatography, and affinity chromatography). These techniques may beused independently or in an appropriate combination to isolate andpurify the polypeptide(s) of the present invention from a culture.

VII. Analysis of ARTS-1 Polypeptide Aminopeptidase Activity

In light of the homology between the polypeptide predicted from ARTS-1gene with aminopeptidase members of the gluzincin zinc metalloproteasefamily, it was determined if ARTS-1 polypeptide had aminopeptidaseactivity, and furthermore, if the terminal aminopeptidase activity wasspecific for a particular amino acid or group of amino acids.

To accomplish this, commercially available amino acid p-nitroanilidesubstrates were incubated with purified GST-ARTS-1 fusion protein for 1hour under conditions of linear enzyme activity over time. The rate ofamide bond hydrolysis was determined by measuring the absorbance of thep-nitroaniline aminopeptidase reaction product at 380 nm. Eachexperimental point was run in triplicate and each determination utilizedsix concentrations of amino acid p-nitroanilide substrate. Kineticconstants were determined by Lineweaver-Burk analysis. Correlationcoefficients for each line generated were greater than 0.997. Theresults of this analysis are shown in Table 3 below. Each amino acidp-nitroaniline substrate tested is shown in the left colurnr, along withits polar/non-polar/acidic/basic nature. V_(max) is a measure of thetheoretical enzyme maximal velocity, K_(m) is the Michaelis-Mentenconstant for each ARTS-1/substrate combination, k_(cat) is the firstorder rate constant (i.e., the turnover number) for the ARTS-1/substratecombination, and k_(cat)/K_(m) is the second order rate constant for theARTS-1/substrate combination (i.e., a measure of overall catalyticefficiency).

As shown in Table 3, recombinant GST-ARTS-1 protein possessed selectiveaminopeptidase activity against non-polar amino acid substrates with afour-fold range of enzyme activity. Isoleucine-pNA was found to be themost favorable amino acid substrate based upon k_(cat)/K_(m)determination, followed by Phe>Gly>Cys>Leu>Met>Ala>Pro>Val. RecombinantGST-ARTS-1 had no activity against either acidic (Asp or Glu) or basic(Arg, His, or Lys) amino acid substrates.

TABLE 3 Specificity and Rates of ARTS-1 Aminopeptidase Activity V_(max)aa-pNA Polarity (pmol/pmol/min) K_(m) (mM) k_(cat) (s⁻¹) × 10⁻²k_(cat)/K_(m) (s⁻¹M⁻¹) Ile Non-polar 5.81 ± 0.87 1.67 ± 0.02  9.68 ±0.15 57.98 Phe Non-polar 5.14 ± 0.04 1.66 ± 0.03  8.57 ± 0.06 51.61 GlyNon-polar 8.67 ± 0.05 3.67 ± 0.03 14.45 ± 0.08 39.37 Cys Non-polar 8.95± 0.31 4.57 ± 0.20 14.92 ± 0.52 32.64 Leu Non-polar 9.45 ± 0.43 5.26 ±0.25 15.75 ± 0.72 29.94 Met Non-polar 13.36 ± 0.75  7.71 ± 0.43 22.27 ±1.25 28.88 Ala Non-polar 26.18 ± 0.24  16.84 ± 0.18  43.63 ± 0.40 25.91Pro Non-polar 5.29 ± 0.08 4.68 ± 0.06  8.82 ± 0.13 18.84 Val Non-polar5.31 ± 0.31 5.69 ± 0.26  8.85 ± 0.52 15.50 Asp Acidic No Activity — NoActivity — Glu Acidic No Activity — No Activity — Arg Basic No Activity— No Activity — His Basic No Activity — No Activity — Lys Basic NoActivity — No Activity —

VIII. Analysis of ARTS-1 TNFR1 Ectodomain Sheddase Regulatory Activity

In light of the ability of the ARTS-1 polypeptide to bind to the TNFR1ectodomain in the yeast two-hybrid interaction assay and thepeptidase/protease motif contained within the predicted polypeptide, theability of ARTS-1 to promote the shedding of the TNFR1 ectodomain fromthe surface of human cells in culture was examined. The methods used inthis experiment are described in detail in Examples 9 and 11. Theresults are depicted in FIGS. 7 and 8.

This experiment was done in two phases. The first phase involvedconstruction of stably transfected cell lines which expressed eitherreduced or elevated levels of ARTS-1 polypeptide, as detailed in Example9. The NCI-H292 cell line was stably transfected with one of threeconstructs, all based on the pTarget vector. The pTarget vector containselements which enable its selection following stable integration in theNCI-H292 cell line, and also has the ability to constitutively express acloned insert in mammalian cells. The pTarget vectors used in thisexperiment were:

1) an empty pTarget vector,

2) pTarget vector containing the fuill length ARTS-1 cDNA coding regionin the sense orientation,

3) pTarget vector containing ARTS-1 cDNA bases 61 to 213 in theanti-sense orientation (this region overlaps the putative transcriptionstart site and intracellular and transmembrane domains).

Following the introduction and selection of these constructs in the hostcell lines, membrane fractions were prepared from the lines and subjectto Western immunoblotting in order to assess ARTS-1 polypeptideexpression. This analysis was conducted according to the Westernimmunoblotting technique described in Example 5 and used the anti-ARTS-1polyclonal antiserum produced as described in Example 4. The results ofthis analysis are shown in FIG. 7. In that Figure, two independentclonal lines containing ARTS-1 sense or antisense expressing vectorswere analyzed. It was found that integration of the empty pTarget vector(Mock) had little effect on endogenous ARTS-1 expression compared tocells that did not contain any stably integrated plasmid (WT). The celllines expressing the fuill length ARTS-1 cDNA in the sense orientation(ARTS-1) showed a significant increase in ARTS-1 protein expression,while the cell lines expressing the ARTS-1 antisense sequence (AS)showed significant reduction in ARTS-1 protein expression.

The amount of TNFR1 ectodomain shedding occurring in each of these celllines is depicted in FIG. 8. The levels of sTNFR1 ectodomain in cellculture supernatants from these cell lines were assayed using acommercially available sandwich-enzyme-linked immunosorbent assay(ELISA) technique (R & D Systems) with a lower limit of detection of 7.8pg/ml. In FIG. 8, results are displayed as the mean of 5 independentexperiments, with accompanying SEM (standard error of the mean). Asshown in the Figure, the cell lines showing increased ARTS-1 proteinexpression (ARTS-1) also showed a significant increase in the amount ofsTNFR1 present in cell culture supernatants as compared to cellstransfected with the empty pTarget vector (Mock). Conversely, cell lineswith decreased ARTS-1 protein expression (AS) showed significantlydecreased levels of sTNFR1 in cell culture supernatants as compared tocells transfected with the empty pTarget vector (Mock).

The degree of TNFR1 ectodomain shedding as a function of ARTS-1 proteinexpression was also assessed indirectly by determining the relativeamounts of membrane-bound TNFR1 fragment in each of the stablytransfected cell lines described above using the Western immunoblottingtechnique described in Example 5. Crude membrane fractions from thestably transfected NCI-H292 cells described in Example 9 were preparedand resolved by SDS-PAGE and analyzed by Western immunoblotting using amurine anti-human TNFR1 monoclonal primary antibody (R & D System) whichdetects the membrane fragment of the TNF receptor. This Western blot isshown in FIG. 11. As shown in FIG. 11, cell lines over-expressing ARTS-1(ARTS-1) demonstrated a decrease in membrane-associated TNFR1 relativeto non-transfected (WT) or control transfected (Mock) cell lines,consistent with an increase in constitutive TNFR1 ectodomain shedding.Conversely, cell lines expressing anti-sense ARTS-1 mRNA (AS)demonstrated an increase in membrane-associated TNFR1 relative tonon-transfected (WT) or control transfected (Mock) cell lines,consistent with a reduction in constitutive TNFR1 ectodomain shedding.

Results from an experiment analyzing the ability of ARTS-1overexpression to potentiate the shedding of TNFR ectodomain from thesurface of NCI-H292 cells in response to PMA stimulation using thesesame cell lines are shown in FIG. 9. Cell lines overexpressing fulllength ARTS-1 mRNA were stimulated with 0.1 μM phorbol 12-myristate13-acetate (PMA), which has previously been shown to upregulate sTNFR1shedding in NCI-H292 cells (Levine et al., Am. J. Respir. Cell Mol.Biol., 14:254–261 [1996]). As indicated in FIG. 9, the cell linecontaining only the empty pTarget vector showed only a modest increasein sTNFR1 shedding following 24 hours of PMA treatment. However, thecell line overexpressing the ARTS-1 cDNA showed a more dramatic increasein sTNFR1 shedding following 24 hours of PMA treatment, increasing fromapproximately 300 pg/ml to 415.3±4.5 pg/ml, increasing from 485±16.9pg/ml to 914.2±9.5 pg/ml

IX. Analysis of TNFR1 Ectodomain Sheddase Regulatory Activity of ARTS-1Catalytic Mutants

The predicted ARTS-1 polypeptide contains a peptidase/protease consensusmotif found in the aminopeptidase family of gluzincin zincmetalloproteases. It was determined if this peptidase/protease catalyticmotif was necessary for the ability of ARTS-1 to promote the shedding ofthe TNFR1 ectodomain. To conduct this experiment, a series of mutantscontaining point mutations predicted to abolish the ARTS-1peptidase/protease activity were constructed. The construction of celllines expressing these mutants was conducted as described in the sectionabove, with experimental details provided in Examples 10 and 11. Resultsof this experiment are depicted in FIG. 10.

The experiment was done in two phases. The first phase involvedconstruction of stably transfected cell lines which expressed eitherwild-type or mutant forms of the ARTS-1 polypeptide. The ARTS-1 mutantsconstructed for this experiment were designed to eliminatemetalloprotease catalytic activity by disrupting the zincmetalloprotease consensus catalytic motif HEXXH(Y)₁₈E (SEQ ID NO:10;consisting of a zinc binding and catalytic site domains). In the ARTS-1polypeptide, this motif is located at H³⁵³ELAH(Y)₁₈E³⁷⁶ (SEQ ID NO:11).Each of the mutations made lies within this domain. These mutations areH353P, E354V, H353P and E354V in combination, and H357V. These mutationshave been previously shown to abolish zinc binding and/or catalytic(enzymatic) activity in proteins containing the motif (Devault et al.,FEBS Lett., 23154–23158 [1988]; Devault et al., J. Biol. Chem.,263:4033–4040 [1988]; Vallee and Auld, FEBS Lett., 257:138–140 [1989];Vallee and Auld, Biochemistry 29:5647–5659 [1990]; and Wang and Cooper,Proc. Natl. Acad. Sci. USA 90:1222–1226 [1993]).

Six cell lines were created by stably transfecting the NCI-H292 cellswith the six constructs, all based on the pTarget expression vector.These constructs (and resulting cell lines) contained:

1) an empty pTarget vector,

2) the ARTS-1 cDNA (WT) coding region,

3) the ARTS-1 cDNA encoding a H353P mutation,

4) the ARTS-1 cDNA encoding a E354V mutation,

5) the ARTS-1 cDNA encoding a H353P and E354V double mutation, and

6) the ARTS-1 cDNA encoding a H357V mutation.

Following the introduction and selection of these constructs in the hostcell lines, the amount of TNFR1 ectodomain shedding occurring in each ofthe lines was determined by measuring the levels of sTNFR1 ectodomain incell culture supernatants using a commercially availablesandwich-enzyme-linked immunosorbent assay (ELISA) technique (R & DSystems) with a lower limit of detection of 7.8 pg/ml. These results aredepicted in FIG. 10 as the mean of five independent experiments, as wellas the SEM (standard error of the mean). As shown in this Figure, thecell line containing the recombinant ARTS-1 cDNA with no mutations(ARTS-1) showed a significantly elevated level of sTNFR1 in the culturesupernatant compared to cell lines containing no integrated DNA (WT) orcontaining the empty pTarget vector (MOCK). Unexpectedly, each of thecell lines containing mutant forms of the ARTS-1 polypeptide also showedelevated levels of sTNFR1 compared to the control lines (i.e., WT andMOCK lines). This experiment demonstrates an unexpected property of thepresent invention, as the peptidase/protease activity of the ARTS-1polypeptide appears not to be required for its sTNFR1 sheddingregulatory activity.

X. Analysis of ARTS-1/TNFR1 Interaction In Vivo

In light of the of the identification of the ARTS-1 gene by the yeasttwo-hybrid interaction screening, the physical association of ARTS-1 andTNFR1 was verified in vivo in a mammalian cell culture system using aco-immunoprecipitation assay.

Crude membrane fractions from cultured NCI-H292 cells were isolated andincubated with murine anti-human TNFR1 monoclonal antibody (R & DSystem) or 1 ml of anti-ARTS-1 antiserum overnight. Following theincubation, the resulting antibody complexes were immunoprecipitatedusing immobilized protein A/G beads (Pierce), and the precipitatedproteins analyzed by Western immunoblotting.

Two different combinations of precipitation and immunoblotting antibodywere used. The results of these immunoprecipitation experiments areshown in FIG. 12. In one experiment (FIG. 12, top panel), the anti-TNFR1antibody was used in the immunoprecipitation (indicated as “IP” in theFigure), and the anti-ARTS-1 antiserum was used as the primary antibodyin the immunoblotting (indicated as “IB” in the Figure). In a secondexperiment (FIG. 12, bottom panel), the antibodies were reversed, wherethe anti-ARTS-1 antiserum was used in the immunoprecipitation, while theanti-TNFR1 antibody was used as the primary antibody in theimmunoblotting.

As shown in FIG. 12, immunoprecipitation of the NCI-H292 cell membraneproteins with an anti-TNFR1 monoclonal antibody resulted in thecoprecipitation of the 100 kDa ARTS-1 species and, conversely,immunoprecipitation with anti-ARTS-1 antiserum coprecipitated the 55 kDaTNFR1. These results indicate an in vivo protein-protein interactionbetween ARTS-1 and TNFR1 proteins.

Similar immunoprecipitation experiments were also performed using thestably-transfected NCI-H292 cell lines described in Example 9. In thisexperiment, the anti-TNFR1 antibody was used to immunoprecipitateprotein from the various membrane protein fractions, and the resultingimmunoprecipitate was examined by Western immunoblotting usinganti-ARTS-1 antiserum as the primary antibody. As shown in FIG. 13,immunoprecipitation using an anti-TNFR1 monoclonal antibody of cellmembrane protein derived from the anti-sense ARTS-1 cell line (AS)showed decreased amounts of ARTS-1 protein as compared tocontrol-transfected (Mock) or non-transfected (WT) cells, consistentwith decreased ARTS-1 protein expression in anti-sense ARTS-1 cells. Noincrease in ARTS-1 protein levels relative to control cell lines wasdetected following immunoprecipitation of ARTS-1 overexpressing celllines with an anti-TNFR1 monoclonal antibody, which likely reflectsincreased TNFR1 shedding related to ARTS-1 over-expression.

XI. Therapeutic Agents to Treat Immune Diseases and Disorders

The present invention provides at least one polypeptide which promotesthe shedding of TNFR1 (i.e., ARTS-1 polypeptide) from the surface ofcultured human cells, a gene encoding the polypeptide, recombinantvectors comprising the gene, host cells comprising the vectors andantibodies directed against the ARTS-1 polypeptide. It is contemplatedthat these compositions will find use as therapeutic agents for thetreatment of TNF-mediated immune diseases. It is contemplated that thetherapeutic agents or the agents for gene therapy of the presentinvention will be administered to a subject orally, parenterally,systemically or locally. It is also contemplated that genes andpolypeptides which are substantially homologous to the ARTS-1 geneprovided by the present invention will also find use in the treatment ofTNF mediated diseases and disorders.

When compositions of the present invention are used as therapeuticagents or agents for gene therapy for immune diseases, it is notintended that the present invention be limited to a particular disease.For example, the gene or polypeptide of the invention may be used, aloneor in combination, to treat inflammatory diseases including, but notlimited to, rheumatoid arthritis, inflammatory bowel disease, septicshock, cachexia, autoimmune disorders, graft-versus-host disease andinsulin resistance.

In some preferred embodiments of the present invention, when thetherapeutic agent of the invention is administered orally, the agent maybe formulated into a tablet, capsule, granule, powder, pill, troche,liquid drops, suspension, emulsion, syrup or the like. Alternatively,the therapeutic agent may be prepared into a dry product which isre-dissolved just before use. In preferred embodiments, when thetherapeutic agent of the invention is administered parenterally, theagent may be formulated for intravenous injection, intramuscularinjection, intraperitoneal injection, subcutaneous injection, as asuppository, etc. Injections are supplied in the form of unit dosageampules or multi-dosage containers. The formulations of the presentinvention may be prepared by conventional methods using appropriateexcipients, fillers, binders, wetting agents, disintegrating agents,lubricating agents, surfactants, dispersants, buffers, preservatives,dissolution aids, antiseptics, flavoring/perfuming agents, analgesics,stabilizers, isotonicity inducing agents, etc. conventionally used inpharmaceutical preparations.

Each of the above-described formulations may contain pharmaceuticallyacceptable carriers or additives. Specific examples of such carriers oradditives include water, pharmaceutically acceptable organic solvents,collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinylpolymers, sodium alginate, water-soluble dextran, sodium carboxymethylamylose, pectin, xanthan gum, gum arabic, casein, gelatin, agar,glycerol, propylene glycol, polyethylene glycol, vaseline, paraffin,stearyl alcohol, stearic acid, serum albumin, mannitol, sorbitol andlactose. One or a plurality of these additives are selected or combinedappropriately depending on the form of the preparation.

The dosage levels of the therapeutic agent of the invention will varydepending on the age of the subject, the route of administration and thefrequency and duration of administration and may be varied over a widerange as suitable for each subject. When an effective amount of thepolypeptide or antibody of the invention is administered in combinationwith an appropriate diluent and a pharmaceutically acceptable carrier,the effective amount of the polypeptide or antibody is in the range from0.01 to 1000 mg/kg per administration, although other amounts arecontemplated, as appropriate. One skilled in the art is capable ofdetermining the therapeutically effective amount appropriate any givencircumstances. In some embodiments, the therapeutic agent isadministered once a day or in several dosages per day for at least oneday.

In some embodiments of the present invention, at least one gene of thepresent invention is used as an agent for therapy for immune diseases ordisorders. When used as a therapeutic agent, the gene(s) may beadministered systemically or locally. The gene(s) may be delivered bydirect application of the nucleic acid to cells or tissues.

In one embodiment, the present invention is used as a gene therapy agentto treat an inflammatory disease or condition. In one embodiment, thegene therapy agent of the present invention is delivered via a viraldelivery system. In an alternative embodiment, the gene therapy agent ofthe present invention involves a non-viral delivery system.

Viral-mediated gene delivery has been shown to be an effective mechanismfor gene delivery for use in gene therapy. Indeed, methods forviral-mediated gene therapy have recently been shown to be effective inhuman and non-human systems (Kordower et al., Science 290:767–773[2000]; Lee et al., Nature 408:483–488 [2000]; Cavazzana-Calvo et al.,Science 288:669–672 [2000]; Kay et al., Nature Genetics 24:257–261[2000]; Amado and Chen, Science 285:674–676 [1999]; Burton et al., Proc.Natl. Acad. Sci. USA 96(22):12725–12730 [1999]; Zhang, Cancer GeneTher., 6(2):113–138 [1999]; Connelly et al., Blood 91(9):3273–3281[1998]; and Connelly et al., Blood 88(10):3846–3853 [1996]). A number ofviruses have been demonstrated to be effective or potentially effectivetools in recombinant gene delivery to subjects, including adenovirus(lentivirus) vectors, adeno-associated virus vectors, herpes virusvectors, vaccinia virus vectors, and retrovirus vectors. In somepreferred embodiments, the recombinant viral vector comprising theARTS-1 gene of the present invention comprises nucleic acid elementsoperably linked for the purpose of transcribing and translating the geneof the invention in cells in a subject. In preferred embodiments, thesenucleic acid elements consist of a nucleotide sequence encoding theARTS-1 polypeptide, and operably linked promoter and enhancer elementsfor expression of the ARTS-1 gene. In some embodiments, thesepromoter/enhancer elements are widely active in all or many cell types,and direct constitutive expression of the gene (e.g., cytomegalovirus(CMV), SV40 or Rous sarcoma virus (RSV) promoter/enhancer sequences). Inalternative embodiments, operably linked promoter/enhancer elements arerestricted in activity to a single cell type or tissue (e.g.,cardiac-specific or liver-specific promoter/enhancers) (Maniatis et al.,Science 236:1237–1245 [1987]; Voss et al., Trends Biochem. Sci., 11:287[1986]). In further embodiments, a promoter/enhancer element thatimparts inducible (i.e., conditional) expression of an operably linkedopen reading frame (e.g., tetracycline inducible or repressiblepromoters) is used. Furthermore, in other embodiments, operably linkednucleotide sequences include sequences directing proper translationinitiation, post-transcriptional splicing/editing, and/orpolyadenylation. In still other embodiments, in addition to containingnucleotide sequences controlling the expression of the ARTS-1 gene, aviral gene therapy vector further contains the necessary nucleotidesequences for in vitro replication and propagation of the virus,production of infective virion particles, and sequences that impartstability of the DNA in a cellular host (although many viral functionsrequire the presence of a “helper virus”). Collectively, such sequencesare sometimes referred to as the viral “backbone.”

Additionally, a genetic sequences of the invention may be enclosed inphospholipid vesicles such as liposomes, and the resultant liposomesadministered to a subject. Liposomes are biodegradable vesiclescontaining an internal aqueous region surrounded by a lipid bilayer.This structure is able to encapsulate materials (e.g., at least one geneof the present invention). By mixing at least one gene of the presentinvention with phospholipid starting material, a liposome-gene complexwill form. Subsequently, when this complex is cultured with cells, thegene(s) in the complex is taken into the cells (i.e., via lipofection).A liposome-gene complex comprising at least one gene of the presentinvention can be administered to a subject, either locally orsystemically. In addition to liposomes, a plasmid encoding the gene(s)of interest may be used.

Alternatively, direct DNA administration, liposome gene transfer, orviral vector, all comprising at least one gene of the present invention,can be used to transfect cells ex vivo (i.e., not within the subject),followed by the transplantation of the recipient cells into the subject.The source of the cells receiving the gene(s) of the invention can becells that have been removed from the subject, or cells from some othersource. Following delivery of the gene(s) of the present invention intothese cells, the cells are then placed into the subject to providetherapeutic value.

In some embodiments of the present invention, methods are contemplatedfor administering gene(s) of the invention locally to tissues viasurgical or injection protocols, as well as systemically, such as byintravenous or intra-arterial administration. Further, an administrationmethod combined with catheter techniques and surgical operations mayalso be employed.

The dosage levels of the agent for delivering the gene(s) of theinvention vary depending on the age, sex and conditions of the subject,the route of administration, the number of times of administration, andthe type of the formulation, among other considerations. One skilled inthe art is capable of determining the therapeutically effective amountappropriate any given circumstances. Usually, it is appropriate toadminister a gene of the invention in an amount of 0.1–100 mg/adultbody/day, although other concentrations are contemplated, asappropriate.

XII. Diagnostic Agents for Immune Diseases and Disorders

It is contemplated that the ARTS-1 gene of the present invention willfind use as a diagnostic marker for TNF signaling activity. Indeed,levels of ARTS-1 mRNA or ARTS-1 polypeptide in biological samples (e.g.,blood, urine, serum or any other body fluid) are usefuil indicators ofthe levels of TNF signaling activity in vivo. It is contemplated thatthe presence of elevated ARTS-1 mRNA and/or polypeptide levels correlatewith decreased TNF signaling activity, while reduced levels of ARTS-1mRNA or polypeptide correlate with increased TNF activity. It iscontemplated that excessive or inadequate TNF activity is indicative ofdisease or pathological states. Thus, the present invention providesmethods and compositions for rapid quantitation of ARTS-1 mRNA andpolypeptide indicative of TNF signaling activity, thereby providinguseful tools for assessing the immune condition of an individualsuspected of suffering from TNF-mediated immune disorders or diseases.In contrast, existing methods for the assay of TNF activity involvelengthy tissue culture assays which are not readily applicable for useas a rapid diagnostic tool in a clinical setting (Suffredini et al., J.Immunol., 155:5038–5045 [1995]; Suffredini et al., N. Eng. J. Med.,321:280–287 [1989]; and Eskandari et al., Immunol. Invest., 19:69–79[1990]).

The present invention further provides compositions for use indiagnostic kits which can be used to rapidly assess ARTS-1 mRNA orpolypeptide using either a nucleic acid probe specific for ARTS-1 mRNA,or PCR primers capable of amplifying ARTS-1 mRNA (all derived from thenucleic acid of SEQ ID NO:1) or the antibody directed against at least aportion of an ARTS-1 polypeptide. Such kits may be designed toincorporate PCR, nucleic acid probe hybridization, and/or antibodyimmunoassay protocols for ARTS-1 marker detection. These kits mayfurther include any reagent(s) or material(s) which makes possible orfacilitates the analysis of a sample (e.g., apparatus for samplecollection, sample tubes, holders, trays, racks, dishes, plates,instructions to the kit user, solutions or other chemical reagents, andsamples to be used for standardization, normalization, and or as controlsamples).

XIII. Identification of Genes Substantially Homologous to ARTS-1

In other embodiments, the present invention provides compositions andmethods for the identification of genes substantially homologous to theARTS-1 gene (i.e., SEQ ID NO:1). It is contemplated that genes similarto the gene set forth in SEQ ID NO:1 have the ability to regulate thecleavage and shedding of TNFR1 ectodomain. In particular, it iscontemplated that the compositions and methods of the present inventionwill find use in stringent hybridization and/or PCR methods to identifygenes substantially homologous to SEQ ID NO:1.

It is further contemplated that genes similar to the gene set forth inSEQ ID NO:1 have the ability to regulate the cleavage and shedding ofthe ectodomains of other pro-inflammatory cytokine receptors (e.g., typeII IL-1 receptor and IL-6 receptor). As discussed above, TNF, IL-1 andIL-6 all are multi-functional pro-inflammatory cytokines which regulateacute phase protein production during innate immune responses toinfection and tissue injury (Suffredini et al., J. Clin. Immunol.,19:203–214 [1999]). Consequently, it is contemplated that genessignificantly homologous to ARTS-1 will find utility in the treatment ofimmune disorders or diseases mediated by abnormal TNF, IL-1 or IL-6activity. It is contemplated that the methods and compositions of thepresent invention will find use in promoting the cleavage and sheddingof the TNFR1 ectodomain, as well as the ectodomains of IL-1 and IL-6cytokine receptors. Although it is not intended that the presentinvention be so limited, two methods for isolation of genessubstantially homologous to the ARTS-1 gene are provided below.

A. Hybridization to Identify Genes Significantly Homologous to theARTS-1 Gene

In this method, a probe derived from the nucleic acid of SEQ ID NO:1 isused to screen a phage EDNA library. The probe is preferably derivedfrom the ARTS-1 gene coding region. The probe may be an oligonucleotideamplified or excised from a larger nucleic acid (e.g., from a purifiedrestriction digest product of a plasmid or other vector) or producedsynthetically, recombinantly or by PCR amplification. There is nolimitation on the size of the probe, although it is preferably longerthan 25 nucleotides, and most preferably encompasses the entire codingregion of the ARTS-1 cDNA. This probe may be single or double stranded.In particularly preferred embodiments the probe is labelled with a“reporter molecule,” so that is detectable in a detection system ofchoice. A detection system may include, but is not limited to, enzymaticdetection, fluorescence, radioactivity, and luminescent systems. Indeed,it is not intended that the present invention be limited to anyparticular detection system or label.

As a source of nucleic acid to be used in the hybridization screening,it is contemplated that nucleic acid from a wide variety of eukaryoticsources may be used. However, λgt10-based EDNA bacteriophage librariesderived from human sources are preferred and advantageous in thisembodiment of the invention. Nonetheless, the source of the nucleic acidto be screened is by no means limited.

Example 1 provides experimental protocols used in the development of thepresent invention, and specifically, a protocol for the screening of aphage plaque library with a radiolabelled DNA probe. This same protocolfinds use in identification of genes which are substantially homologousto SEQ ID NO:1. Briefly, a bacteriophage cDNA library is plated in alawn of DH-5α host bacteria. The DNA contained in each of the plaques isreplica plated via a plaque-lift onto a membrane suitable for subsequenthybridization. The DNA is fixed to the filter, and probed in solutionusing the ARTS-1 derived probe under stringent conditions. Phage plaqueDNA with the ability to hybridize to the probe is isolated, analyzed andsequenced.

B. PCR to Identify Genes Significantly Homologous to the ARTS-1 Gene

PCR may be used to identify genes significantly homologous to the ARTS-1gene. In a preferred embodiment, a sense primer and an anti-sense primerderived from the polypeptide coding region of the ARTS-1 gene aresynthesized and used in a polymerase chain reaction (PCR). In analternative embodiment, “degenerate” PCR primers are used. In theseembodiments, the PCR primers have nucleotide sequences which encodeamino acid domains of the ARTS-1 polypeptide, but differ from the ARTS-1gene nucleotide sequence. Criteria for designing “degenerate” PCRprimers are well known in the art. It is not intended that the presentinvention be limited to any particular primer or primer set.

It is also contemplated that nucleic acid from a wide variety ofeukaryotic sources may be used as template in the PCR methods. It is notintended that the present invention be limited as to the source of thetemplate nucleic acid. However, nucleic acid template derived from humansources is the most preferred embodiment of the present invention.

In preferred PCR methods, a nucleic acid that is substantiallyhomologous to the ARTS-1 gene is amplified using materials and methodsknown in the art. Conditions of the PCR may be manipulated (e.g., theduration or temperature of the cycle times may be changed) to amplify anucleic acid substantially homologous to the ARTS-1 gene. Indeed, it iscontemplated that many suitable PCR conditions as known in the art willfind use in the present invention. The nucleic acid amplified in the PCRreaction is visualized, isolated, subeloned, and sequenced usingtechniques standard in the art.

The isolated nucleic acid obtained by either plaque hybridization or PCRis further examined to determine if the isolated nucleic acid is“substantially homologous” to the ARTS-1 gene of the present inventionbased on criteria previously discussed. In a preferred embodiment, theisolated nucleic acid which is substantially homologous to the ARTS-1gene encodes a polypeptide having the ability to promote the cleavageand shedding of at least one of the ectodomains of the group of cytokinereceptors consisting of TNFR1, and IL-1 and IL-6 cytokine receptors. Thenucleic acid obtained is then tested using methods such as thosedescribed in Examples 7, 11, 12 and 13. In a most preferred embodiment,the isolated gene has TNF regulatory activity, as determined using theprotocol supplied in Example 14.

XIV. Methods for Drug Screening

It is contemplated that compounds which are able to regulate ARTS-1activity or receptor ectodomain shedding are candidates for furtherdevelopment as therapeutically advantageous drugs. Such drugs find useas immune response modifiers, to either enhance or attenuate the actionof an cytokine signalling (e.g., TNF, IL-1 or IL-6). The presentinvention provides compositions and methods for the screening andidentification of test compounds which can regulate ARTS-1 activity,ARTS-1 transcript or protein expression, and receptor shedding, andthus, identifies drug candidates for further development astherapeutics. However, an understanding of the mechanism(s) of how aparticular compound regulates cytokine signalling and proinflammatoryimmune responses is not necessary in order to use the present invention.

A. Method for Drug Screening to Identify Compounds Having the Ability toRegulate ARTS-1 Expression

The present invention provides screening methods to identify compoundswhich can regulate the level of ARTS-1 transcript or protein in atissue. Test compounds which are able to regulate ARTS-1 transcript orprotein levels in a cell or tissue are candidates for furtherdevelopment as therapeutic agents. It is contemplated that compoundswhich can upregulate or downregulate ARTS-1 expression also have theability to downregulate or upregulate cytokine signalling (i.e., aproinflammatory immune response), respectively.

In one embodiment of the present invention, the screening method usesNorthern blotting to assess the levels of the ARTS-1 transcript in acell culture following exposure of the culture to a test compound. Inthis embodiment, cultured cells are exposed to a test compound, andsamples of tissue are collected at intervals ranging from approximately0 to 48 hours. RNA is isolated from these cells and subjected toNorthern blot analysis, as described in Example 3, using a probespecific for the ARTS-1 mRNA. Comparison of the ARTS-1 transcript levelsbefore and after exposure to the test compound identifies thosecompounds that upregulate or downregulate ARTS-1 transcript levels. Itis contemplated that compounds that can regulate ARTS-1 transcriptlevels provide targets for further development as therapeutic agents.

In a preferred embodiment, cultured human NCI-H292 pulmonarymucoepidermoid carcinoma cells are used in the screening. However, it isnot intended that the invention be limited to the use of only this celltype, as other cell types are equally suitable, and are known to thosein the art. Similarly, it is not intended that the reagents, methods orapparatus for RNA collection and analysis be limited to those describedherein, as numerous suitable equivalents are known to those in the art.

In another embodiment of the screening method of the present invention,Western immunoblotting is used to assess the levels of ARTS-1 proteinfollowing exposure of a cell culture to a test compound. In thisembodiment, cultured cells were exposed to a test compound, in thiscase, 4b-phorbol 12-myristate 13-acetate (PMA), and samples of tissuewere collected at 0, 2, 8 and 24 hours following the exposure. Membraneproteins were isolated from these cells and subjected to Westernimmunoblot analysis, as described in Examples 4 and 5, using antiserumspecific for the ARTS-1 protein. Results of this screening are shown inFIG. 14, Panel A. Comparison of the ARTS-1 protein levels in the cellsbefore and after exposure to the test compound demonstrated thattreatment of the cells with PMA resulted in an upregulation of ARTS-1protein expression. It is contemplated that compounds that canupregulate ARTS-1 protein levels are therapeutically advantageous, assuch compounds can suppress a proinflammatory immune response byenhancing receptor ectodomain shedding. However, an understanding of themechanism(s) is not required in order to use the present invention. Asindicated herein, PMA is a candidate for further development as atherapeutic agent.

In an alternative preferred embodiment, cultured human NCI-H292pulmonary mucoepidermoid carcinoma cells are used in the screening.However, it is not intended that the invention be limited to the use ofonly this cell type, as other cell types are equally suitable, and areknown to those in the art. Similarly, it is not intended that thereagents, methods or apparatus for protein collection and Westernimmunoblot analysis be limited to those described herein, as numeroussuitable equivalents are known to those in the art.

B. Method for Drug Screening to Identify Compounds Having the Ability toEnhance Receptor Ectodomain Shedding

The present invention provides screening methods to identify compoundswhich can regulate receptor ectodomain shedding. Test compounds whichare able to regulate ectodomain shedding are identified as candidatesfor further development as therapeutic agents. It is contemplated thatcompounds which can upregulate or downregulate ectodomain shedding alsohave the ability to downregulate or upregulate a proinflanunatory immuneresponse, respectively.

In one embodiment of the present invention, the screening method uses anELISA to assess the levels of sTNFR1 in the supernatant of cellcultures. In the experiments described herein, an ELISA was used toassess the levels of sTNFR1 in the supernatants from human NCI-H292pulmonary mucoepidermoid carcinoma cells following exposure of theculture to PMA. In this embodiment, the cultured cells were exposed tothe compound, and samples of supernatant were collected at 0, 2, 8 and24 hours following the exposure. The samples were analyzed using anELISA with an anti-sTNFR1 antibody as described in Example 11. Theresults of this ELISA screening are shown in FIG. 14, Panel B. As can beseen in the Figure, treatment of the cultured cells resulted in anincrease in sTNFR1 shedding over the course of 24 hours compared to acontrol culture (n=5,*P<0.05). Thus, this screening method identified acompound as a candidate for further drug development.

It is contemplated that compounds that can upregulate sTNFR1 ectodomainshedding are therapeutically advantageous, as such compounds cansuppress a proinflammatory immune response. However, an understanding ofthe mechanism(s) is not required in order to use the invention. Based onthe criteria set forth and described herein, PMA is a candidate forfurther development as a therapeutic agent.

It is not intended that the method for drug screening assessing sTNFR1ectodomain shedding be limited to analysis of sTNFR1 shedding. It iscontemplated that the analysis of ectodomain shedding other cytokinereceptors, such as type II interleukin-1 cytokine receptor andinterleukin-6 cytokine receptor alpha-chain gp80 also find use with thepresent invention.

C. Method for Drug Screening to Identify Compounds Having the Ability toRegulate the Peptidase Activity of ARTS-1 Protein

The present invention provides screening methods to identify compoundswhich can regulate the peptidase activity of ARTS-1 protein. Testcompounds which are able to regulate ARTS-1 peptidase activity arecandidates for further development as therapeutic agents. It iscontemplated that compounds which can upregulate or downregulate ARTS-1peptidase activity also have the ability to downregulate or upregulate aproinflammatory immune response, respectively.

In one embodiment of the present invention, the screening method usesamino acid p-nitroanilide substrates to assess the aminopeptidaseactivity (described in Example 7) of purified recombinant GST-ARTS-1fusion protein (described in Example 6). In this screening method, theamino acid p-nitroanilide hydrolysis reaction is conducted in theabsence and presence of a test compound, according to the reactionconditions provided in Example 7, and the rate of amide bond hydrolysisby ARTS-1 is determined. It is then observed whether a test compound hasthe ability to regulate the rate of amide bond hydrolysis.

It is contemplated that compounds that can regulate ARTS-1 amide bondhydrolysis are therapeutically advantageous, as such compounds canregulate a proinflammatory immune response, and are targets for furtherdevelopment as therapeutic agents. However, an understanding of themechanism(s) is not required in order to use the present invention.

It is not intended that this method for drug screening be limited tothose reagents itemized in Example 7. For example, it is contemplatedthat purified ARTS-1 proteins in addition to ARTS-1-GST also find usewith the present invention. Furthermore, it is contemplated that morethan one amino acid p-nitroanilide substrate finds use with thescreening method, as isoleucine p-nitroanilide, phenylalaninep-nitroanilide and glycine p-nitroanilide substrates can all be used inthe hydrolysis reaction.

Experimental

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); l or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); Accurate Chemical and Scientific Corporation(Accurate Chemical and Scientific Corporation, Westbury, N.Y.); AdvancedBiotechnologies (Advanced Biotechnologies Incorporated, Columbia, Md.);Amersham/Pharmacia (Amersham/Pharmacia Biotech, Piscataway, N.J.); ATCC(American Type Culture Collection, Rockville, Md.); Bachem (Bachem, Kingof Prussia, Pa.); Biofluids (Biofluids, Inc, Rockville, Md.); BoehringerMannheim (Roche/Boehringer Mannheim Corporation, Indianapolis, Ind.);Calbiochem (Calbiochem-Novabiochem Corp, San Diego, Calif.); Clontech(Clontech Laboratories, Inc., Palo Alto, Calif.); Genzyme (GenzymeCorporation, Cambridge, Mass.); Life Technologies (LifeTechnologies/Gibco/BRL, Gaithersburg, Md.); Novex (Novex/Invitrogen,Carlsbad, Calif.); Pierce (Pierce, Rockford, Ill.); Promega (PromegaCorporation, Madison, Wis.); R & D Systems (R & D Systems, Minneapolis,Minn.); Research Genetics (Research Genetics, Huntsville, Ala.); Roche(Roche/Boehringer Mannheim Corporation, Indianapolis, Ind.); Sigma(Sigma Chemical Co., St. Louis, Mo.); Stratagene (Stratagene, La Jolla,Calif.).

The following Examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

EXAMPLE 1 Identification of ARTS-1, a Novel Human AminopeptidaseRegulator of Type1 Tumor Necrosis Factor Receptor (TNFR1) Shedding

A) Yeast Two-Hybrid Library Screening

A yeast two-hybrid library screening was conducted to identify proteinsthat interact with or are capable of interacting with the extracellulardomain of the type I, 55 kDa TNF receptor (TNFR1). A human lung cDNAlibrary (Clontech), cloned into pGAD10 (GAL4 DNA-activation domainvector), was screened to detect interactions with a GAL4BD-TNFR1 fusionprotein using a Matchmaker System 2 (Clontech) using methods known inthe art as well as the manufacturer's recommended protocols. Amino acids26 to 216 of TNFR1, corresponding to the extracellular domain (i.e., theectodomain) located between the putative leader domain and thetransmembrane domain (Nophar et al., EMBO J., 10:3269–3278 [1990]) werecloned into pAS2-1 (GAL4 DNA-binding domain vector) to generate the baitGAL4 DNA-binding domain fusion protein, GAL4BD-TNFR1. The Y190 yeaststrain was transformed with the pAS2-1 GAL4BD-TNFR1 bait plasmid by thelithium acetate method utilizing the Yeastmaker Transformation System(Clontech). Transformed yeast were selected on synthetic drop-platesdeficient in tryptophan, leucine and histidine in the presence of 25 mM3 amino-1,2,4-triazole (SIGMA). His⁺ colonies were then subjected toβ-galactosidase colony-lift filter assays and β-galactosidase producingcolonies were selected and restreaked on synthetic drop-out plates.Approximately 7.11×10⁶ transformants were screened. β-galactosidasepositive colonies which produced a blue signal within 2 hours wereselected for further study. Selected colonies were analyzed forexpression of the GAL4 binding domain (GAL4BD) fusion protein byimmunoblotting using an anti-GAL4BD monoclonal antibody (Clontech).Thirty three positive clones were identified which activated theβ-galactosidase reporter construct. All 33 positive clones weresequenced using an ABI Perkin Elmer 377 automated fluorescent sequencer.

One positive clone, L26C-53A, encoding a consensus zinc metalloproteasecatalytic motif was selected for further study. This clone contained a2355-bp insert containing an open reading frame of 631 amino acids, butdid not contain a poly(A) tail or a putative translation initiationsite. The gene encoded by this insert was entitled aminopeptidaseregulator of type-1, 55 kDa tumor necrosis factor receptor shedding (orARTS-1). The L26C-53A sequence corresponds to ARTS-1 bases 1044 to 3082.

B) ARTS-1 cDNA Cloning/Phage Plaque Hybridization

Using ³²P-labelled DNA derived from clone L26C-53A as a probe, aPMA-stimulated NCI-H292 cell line cDNA library (Stratagene) was screenedto obtain ARTS-1 cDNA clones. The NCI-H292 cell line was derived fromhuman pulmonary mucoepidermoid carcinoma cells and has been demonstratedto shed cell surface sTNFR1 in response to PMA stimulation (Levine etal., Am. J. Respir. Cell Mol. Biol., 14:254–261 [1996]). The uni-ZAP XRphage cDNA library (Stratagene) was constructed with NCI-H292 cell poly(A⁺) mRNA isolated following 24 hours of stimulation with 1 μM PMA(phorbol 12-myristate, 13-acetate, Sigma).

Bacteriophage from the uni-ZAP library were plated in a lawn of XL1-Blue(MRF-) E. coli (Stratagene) at a density of 50,000 pfu per 150 mm plateand incubated overnight at 37° C. Plaques were transferred to Hybond N+filters (Amersham/Pharmacia) and denatured for 5 minutes in 1.5M sodiumchloride, 0.5 M sodium hydroxide. Filters were then neutralized in twowashes of 5 minutes each in 1.5M sodium chloride, 1M Tris-base, rinsedin 3×SSC and UW cross-linked. Filters were pre-hybridized in10×Denhardt's solution, 6×SSPE, 1% SDS at 42° C. for 4 hours inhybridization buffer. Filters were hybridized overnight at 42° C. withapproximately 10⁷ CPM of ³²P-labelled L26C-53A insert which wasgenerated by random priming. Filters were then washed in 2×SSC, 0.5% SDSfor 20 minutes at room temperature, followed by two 1 hour washes in1×SSC, 0.1% SDS at 65° C. Filters were then exposed to x-ray filmovernight and positive plaques were selected. Positive plaques weresubjected to two additional rounds of plaque hybridization prior tosequencing. Positive plaques were recovered via in vivo excisionutilizing the ExAssist helper phage (Stratagene).

Following 3 rounds of screening, four hybridizing phage clones wereidentified, then sequenced utilizing the ThermoSequenase cyclesequencing kit (Amersham/Pharmacia). These four hybridizing clonesoverlapped the L26C-53A sequence, but none encoded a full-length cDNA.Sequencing revealed one phage clone (bp 1–1777) which contained theputative 5′ UTR and three phage clones which contained the putative 3′UTR and the poly(A) tail (bp 2181–4845). cDNA sequence encoding theportion of the gene lying between these 5′ and 3′ terminal clones wasobtained via PCR from Marathon-Ready™ human lung cDNA (Clontech)utilizing Pfu Turbo DNA Polymerase (Stratagene) and the followingprimers:

(SEQ ID NO:3) 5′-ATAACCATCACAGTGAGGGGGAGG-3′ (1684–2279) and (SEQ IDNO:4) 5′-TAGTTGACTCCGCAGCATTCGCTC-3′ (2257–2280)The cDNA segment amplified with these primers was cloned into pGemTEasy(Promega) and both strands were sequenced by automated fluorescentsequencing using an ABI Perkin Elmer 377 automated fluorescentsequencer. Sequences of all overlapping regions from the originaltwo-hybrid clone L26C-53A (bp 1044 to 3082), the four NCI-H292 cDNAlibrary clones and the PCR product (bp 1684 to 2280) showed nodiscrepancies in the nucleotide sequence in their overlapping regions.

EXAMPLE 2 Molecular Analysis of the ARTS-1 cDNA and PredictedPolypeptide

The complete cDNA corresponding to the human ARTS-1 transcription unitcontains 4845 nucleotides and encodes an open reading frame of 2823 bp,as shown in FIG. 1 and set forth in SEQ ID NO:1. The first in-frame ATGcodon, located at nucleotide 88, follows an in-frame stop codon, locatedat nucleotide 76, and matches the −3 and +4 nucleotides of the consensusKozak sequence consistent with a strong initiator codon (aagatgg)(Kozak, J. Cell Biol., 108:229–241 [1989]). Sequence analysis revealedfive potential N-glyeosylation sites and a TAA stop codon, located atnucleotide 2911. The putative 3′ untranslated region contained aconsensus polyadenylation site (AATAAA), located at nucleotides 4876 to4800, which is 18 nucleotides upstream of a 27 nucleotide poly(A) tail.The 3′ untranslated region also included two ATTTA sites, located atnucleotides 3929 and 4457; this sites have been identified as an mRNAdestabilization motif in mRNA's for cytokines, oncogenes andtranscriptional activating factors (Stephens et al., J. Biol. Chem.,267:8336–8341 [1992]).

The open reading frame predicted from the human ARTS-1 cDNA encodes aprotein of 941 amino acid residues (See, FIG. 1 and SEQ ID NO:2) with acalculated molecular weight of 107,227 Da and an estimated pI of 6.0.Sequence analysis, including Kyte-Doolittle hydropathy prediction (Kyteand Doolittle, J. Mol. Biol., 157:105–132 [1982]) was performed usingMacVector 7.0 software (Oxford Molecular). The location of the putativehydrophobic transmembrane α-helical domain was predicted utilizingseveral web-based analysis programs (MEMSAT2 (McGuffin et al.,Bioinform., 16:404–405 [2000]; Sosui (Hirokawa et al., Bioinform.,14:378–379 [1998]; TMAP (Persson and Argos, J. Mol. Biol., 237:182–192[1994]); TMpred (Hofmann and Stoffel, Biol. Chem. Hoppe-Seyler 374:166[1993]; and TopPred2 (von Heijne, J. Mol. Biol., 225:487–494 [1992]). Insum, ARTS-1 is predicted to be a type II integral membrane protein witha single hydrophobic transmembrane α-helical domain, located betweenamino acids 5 and 28 (See, FIGS. 1 and 3), and a very short hydrophobicintracellular amino-terminal domain (See, McGuffin et al., Bioinform.,16:404–405 [2000]; Hirokawa et al., Bioinform., 14:378–379 [1998];Persson and Argos, J. Mol. Biol., 237:182–192 [1994]); Hofmann andStoffel, Biol. Chem. Hoppe-Seyler 374:166 [1993]; and von Heijne, J.Mol. Biol., 225:487–494 [1992]).

The protein sequence analysis using MacVector software revealed a zincmetalloprotease consensus catalytic motif (HEXXH(Y)₁₈E) (SEQ ID NO:10),which is predictive of an active metalloprotease (Hooper, ZincMetalloproteases in Health and Disease, Taylor & Francis, London,England [1996], p. 1–21). Utilizing various web-based analysis programs,a putative amino acid transmembrane domain (as predicted by von Heijne,J. Mol. Biol., 20:487–494 [1992]), extending from amino acids 5 to 28,was identified. These features suggest that human ARTS-1 is a type IIintegral membrane protein with a single hydrophobic transmembraneα-helical domain, located between amino acids 5 and 28 (See, FIGS. 1 and2), and a very short, hydrophobic intracellular amino-terminal domain(See, McGuffin et al., Bioinform., 16:404–405 [2000]; Hirokawa et al.,Bioinform., 14:378–379 [1998]; Persson and Argos, J. Mol. Biol.,237:182–192 [1994]); Hofinann and Stoffel, Biol. Chem. Hoppe-Seyler374:166 [1993]; and von Heijne, J. Mol. Biol., 225:487–494 [1992]).

A BLAST protein homology comparison revealed that the human ARTS-1contained significant homology with several members of theaminopeptidase family of gluzincin zinc metalloproteases (See, Tables 1and 2, supra) (Wang and Cooper, Zinc Metalloproteases in Health andDisease, Taylor & Francis, London, England [1996]). As has previouslybeen reported for other aminopeptidase family members, the human ARTS-1was found to contain a 375 amino acid domain that is highly conservedwith human placental leucine aminopeptidase (PLAP) (Rogi et al., J.Biol. Chem., 271:56–61 [1996]), rat insulin-regulated aminopeptidase(IRAP) (Keller et al., J. Biol. Chem., 270:23612–23618 [1995]), humanaminopeptidase A (AMP A) (Nanus et al., Proc. Natl. Acad. Sci. USA90:7069–7073 [1993]; Li et al., Genomics 17:657–664 [1993]), humanaminopeptidase N (AMP N) (Olsen et al., FEBS Lett., 238:307–314 [1988]),human puromycin sensitive aminopeptidase (PSA) (Tobler et al., J.Neurochem., 68:889–897 [1997]), rat thyrotropin-releasing hormonedegrading enzyme (TRH DE) (Schauder et al., Proc. Natl. Acad. Sci. USA91:9534–9538 [1994]), Saccharomyces cerevisiae aminopeptidase YSCII(Garcia-Alvarez et al., Eur. J. Biochem., 202:993–1002 [1991]), C.elegans cosmid F49E8.3 gene product (Wilson et al., Nature 368:32–38[1994]), and Lactococcus lactis aminopeptidase N (Tan et al., FEBSLett., 306:9–16 [1992]). This highly conserved domain contains theputative consensus zinc binding domain and catalytic site(T³⁵⁰VAHELAHQWFG (SEQ ID NO:8) and L³⁷²WLNEGFA (SEQ ID NO:9) which ischaracteristic of aminopeptidase family members (Wang and Cooper, ZincMetalloproteases in Health and Disease, Taylor & Francis, London,England [1996]; Keller et al., J. Biol. Chem., 270:23612–23618 [1995]).Furthermore, PLAP, AMP A, AMP N, IRAP and TRH DE share structuralsimilarity with human ARTS-1, based upon the presence of a transmembranedomain and a short intracytoplasmic tail, consistent with a type IIintegral membrane protein.

EXAMPLE 3 ARTS-1 mRNA Expression Analysis

Following analysis of the ARTS-1 cDNA, the expression pattern ofendogenous of ARTS-1 mRNA was investigated using a Northernimmunoblotting protocol. RT-PCR was performed on human lung poly(A⁺)mRNA (Clontech) to generate the human ARTS-1 cDNA coding sequenceutilizing the following primers:

(SEQ ID NO:5) 5′-GCAAGAAGATGGTGTTTCTGCCCCTC-3′ (80–105) and (SEQ IDNO:6) 5′-TTACATACGTTCAAGCTTTTCACT-3′ (2890–2913).The full length ARTS-1 coding sequence amplified by these primers wascloned into the pTarget mammalian expression vector (Promega). The DNAsequence of this cloned open reading frame was obtained from bothstrands by automated fluorescent sequencing, as known in the art. Therewere no discrepancies between the sequence obtained from these twostrands, nor from the sequence obtained from the PMA-stimulated NCI-H292cell line cDNA library. A ³²P-labelled cDNA probe, corresponding to thefull length ARTS-1 coding sequence, was utilized as a probe for Northernblotting of human multiple tissue Northern blots (Clontech) according tothe manufacturer's protocol.

As shown in FIG. 3, Northern blot analysis utilizing poly(A+) mRNA frommultiple human tissues revealed that the human ARTS-1 transcript wasexpressed in multiple tissues, including spleen, thymus, small and largeintestine, peripheral blood leukocyte, heart, placenta, lung, skeletalmuscle, kidney and pancreas. In these tissues, an approximately 5.7 kBpredominant mRNA species was detected (See, FIG. 3, top panel). Alsoshown is the same blot following stripping and rehybridization to aprobe specific for the human GAPDH transcript as a reference for RNAloading normalization (See, FIG. 3, bottom panel).

EXAMPLE 4 The Generation of Polyclonal Anti-ARTS-1 Antiserum

In order to conduct studies of the ARTS-1 polypeptide, polyclonalantiserum was generated against the ARTS-1 polypeptide. Specifically, a17 amino acid ARTS-1 synthetic peptide was used to immunogenize NewZealand white rabbits and subsequently collect immune serum using acommercial service (Research Genetics). This peptide corresponded toamino acids 538 to 554 of the ARTS-1 protein and had the sequence:

-   -   R⁵³⁸GRNVHMKQEHYMKGSD⁵⁵⁴ (SEQ ID NO:7)        This particular peptide was chosen based upon its antigenic        potential and its lack of homology with other protein sequences        via BLAST homology search.

Rabbits were immunized with this peptide using standard techniques. TheARTS-1 peptide was conjugated to KLH and mixed with an equal volume ofFreund's complete adjuvant. The amount of antigen utilized perimmunization was 0.1 mg, which was injected into three subcutaneousdorsal sites. The animals received boosts at weeks 2, 6, and 8. Bleedswere obtained at weeks 4, 8 and 10 and tested for the presence ofanti-ARTS-1 antibody. In subsequent experiments, the antiserum obtainedfrom the 10 week bleed was used.

EXAMPLE 5 Analysis of ARTS-1 Polypeptide Expression in Cultured CellLines and Primary Cells

Following the production of ARTS-1 polyclonal antiserum, the expressionof endogenous ARTS-1 polypeptide in cultured cell lines and primarycells was investigated using standard Western immunoblotting as known inthe art. Protein concentrations were determined via the BCA proteinassay (Pierce). Following protein quantitation, 20 microgram samples ofprotein were boiled for 5 minutes in Laemmli buffer. Samples were thenresolved via SDS-PAGE (6% polyacrylamide) and electroblotted ontonitrocellulose (Novex). Blots were incubated overnight in blockingbuffer (5% wt/vol nonfat dry milk in PBS/0.1% Tween-20), then incubatedfor 2 hours with ARTS-1 antiserum at a 1:20,000 dilution in blockingbuffer. Membranes were washed three times for 5 minutes each wash inPBS/0.1% Tween; incubated for 2 hours with 0.8 mg/ml horseradishperoxidase conjugated goat anti-rabbit IgG (Life Technologies) dilutedto 1:5,000 in blocking buffer, then washed three times for 5 minuteseach wash in PBS/0.1% Tween and finally washed three times for 5 minuteseach wash in PBS/0.3% Tween. Membranes were then incubated inchemiluminescent detection substrate for 1 minute and signal detected onX-ray film.

Crude homogenates made from NCI-H292 cells for immunoblot analysis wereproduced by cell lysis in homogenization buffer consisting of 200 μl of50 mM Tris-HCl, pH 7.2, containing 0.1% Triton X-100 and Complete™protease inhibitor cocktail tablet (Boehringer Mannheim) and sonicatedfor four times at 15 seconds each using a microprobe. The homogenate wascentrifuged at 1,000×g for 5 minutes to remove nuclei, unbroken cellsand debris. The low speed supernatant was either utilized as a wholecell lysate or was further fractionated by ultracentrifugation at100,000×g for 1 hour to generate a crude cytosolic and membranefractions. The crude membrane fraction was resolubilized inhomogenization buffer prior to immunoblotting.

Specificity of the resulting polyclonal antiserum was first tested usingcrude whole cell homogenates, and membrane and cytosolic fractionsprepared from cultured NCI-H292 cells. These results are shown in FIG.4. Comparing the top two panels of FIG. 4, the antiserum was shown todetect a predominant 100 kDa membrane form and a predominant 68 kDacytosolic form from the NCI-H292 cells. The whole cell extracts revealeda mixture of these two forms. The preimmune serum showed no reactivitytowards the same samples when used at the same concentration.

Specificity of the immune serum was further demonstrated in competitionexperiments as shown in the bottom two panels of FIG. 4. In thesecompetition experiments, 1 μl of the ARTS-1 antiserum was pre-incubatedwith 1 mg of either bovine serum albumin or RGRNVHMKQEHYMKGSD peptide(SEQ ID NO:7) for two hours prior to utilization for immunoblotting.Preincubation of the immune serum with the peptide against which thepolyclonal antiserum was raised resulted in almost complete attenuationof the immune signal (Bottom panel). In contrast, preincubation of theimmune serum with bovine serum albumin resulted in minimal attenuationof immune signal (Third panel).

The expression of endogenous ARTS-1 polypeptide in primary cells andother cell lines was further investigated using the anti-ARTS-1antiserum and Western immunoblot technique, as shown in FIG. 5. Theseexperiments analyzed membrane and cytosolic fractions made from humanbronchial brushing specimens, the airway epithelial cell lines BEAS-2Band BET-1A, human lung carcinoma cell line A549, cultured NCI-H292cells, primary cultures of normal human bronchial epithelial cells(NHBE), human umbilical vein endothelial cells (HUVEC) and humanfibroblasts. These experiments also revealed multiple sized forms ofARTS-1 polypeptide on the Western immunoblot, including 132, 100 and 68kDa forms which may localize to various subcellular fractions. Thesemultiple sized forms may be due to regulated processing of the ARTS-1polypeptide, and furthermore may indicate that this processing iscompartmentalized.

EXAMPLE 6 Expression and Purification of Recombinant GST-ARTS-1Polypeptide

The cDNA sequence of the ARTS-1 extracellular domain (i.e., theectodomain; amino acids 30–941) was PCR amplified from the pTargetplasmid containing the full length ARTS-1 coding sequence. The ARTS-1extracellular domain was then cloned into the pGEX-6P-1 plasmid(Amersham/Pharmacia) and used to transform the BL21 E. coli host strain.Colonies expressing GST-ARTS-1 fusion protein were selected by Westernblotting utilizing an anti-GST antibody (Amersham/Pharmacia). Positiveclones were grown at room temperature, stimulated for 4 hours with 0.6mM isopropyl β-D-thiogalactoside (IPTG) and subsequently lysed withB-PER protein extraction reagent (Pierce). Cells were centrifuged at27,000×g for 15 minutes to separate the soluble from the insolublefractions. Following treatment with 0.2 mg/ml lysozyme for 5 minutes,the GST-ARTS-1 fusion protein was isolated from the insoluble fractionby denaturation with 6M urea in PBS. The GST-ARTS-1 fusion protein wasrefolded by serial dialysis against PBS baths containing decreasing ureaconcentrations (5 M to 0 M). The GST-ARTS-1 fusion protein was purifiedutilizing a glutathione sepharose 4B affinity column and eluted withreduced glutathione buffer using techniques known in the art.

To assess the purity of the eluted recombinant GST-ARTS-1 fusionprotein, samples were subjected to SDS-PAGE on a 4%–12% gradient gel(Novex) and stained with Coomassie brilliant blue. FIG. 6A shows theresult of this experiment. In this Figure, soluble and insoluble proteinfractions from BL21 E. coli transformed with empty pGEX-6P-1 vector(lanes 1 and 2) or the pGEX-6P-1-ARTS-1 vector (lanes 3 and 4) areshown. The GST-ARTS-1 fusion protein following elution is shown in lane5 as a predominant 132 kDa band, corresponding to the predictedmolecular weight of a GST-ARTS-1 fusion protein (104 kDa ARTS-1extracellular domain plus 26 kDa GST tag). The control purified GST tagis revealed as a predicted 26 kDa band in lane 6.

Purified recombinant GST-ARTS-1 fusion protein samples were furthersubjected to FPLC analysis (LKB LCC-500 plus, Amersham/Pharmacia)utilizing a Superose 6 HR 10/30 gel filtration column(Amersham/Pharmacia). Recombinant purified GST-ARTS-1 fusion proteinsamples were eluted with PBS at a flow rate of 0.5 ml/min and fractionswere collected every 1 minute. Absorbance was recorded at 280 mn with achart speed of 0.25 cm/min. FIG. 6B shows the result of this analysis.In this figure, FPLC analysis of the purified GST-ARTS-1 revealed asingle major protein elution peak which eluted at approximately 40minutes.

Using the aminopeptidase activity assay described below, FPLC fractionswere assessed for aminopeptidase activity utilizing a phenylalaninep-nitroaniline substrate. As shown in FIG. 6C, the single major proteinpeak revealed by FPLC analysis coeluted with a peak of aminopeptidaseactivity against a phenylalanine p-nitroaniline substrate in pooledfractions from 38–44 minutes.

EXAMPLE 7 ARTS-1 Aminopeptidase Activity Assay

Aminopeptidase activity of the recombinant GST-ARTS-1 fusion protein wasassessed by determination of the rate of amide bond hydrolysis of aminoacid p-nitroanilide substrates (Bachem). Amino acid p-nitroanilides(final concentrations 0.25 to 8 mM) were incubated at room temperaturewith 24 pmoles of GST-ARTS-1 fusion protein in 200 μl of 50 mM Tris, pH7.5 for 1 hour. Reactions were terminated by addition of 280 μl of 3Msodium acetate (pH 5.2). The rate of amide bond hydrolysis wasdetermined by measuring the absorbance of p-nitroaniline at 380 nm (See,Table 3, supra). Spontaneous hydrolysis of the substrate was correctedfor by subtracting the absorbance of control incubations which wereterminated immediately. Kinetic constants were determined byLineweaver-Burk analysis. Each experimental point was assayed intriplicate and each determination utilized six concentrations of eachamino acid p-nitroanilide substrate. Correlation coefficients for eachline generated were greater than 0.997.

As shown in Table 3, recombinant GST-ARTS-1 protein possessed selectiveaminopeptidase activity against non-polar amino acid substrates with afour-fold range of enzyme activity. Isoleucine-pNA was found to be themost favorable amino acid substrate based upon k_(cat)/K_(m)determination, followed by Phe>Gly>Cys>Leu>Met>Ala>Pro>Val. RecombinantGST-ARTS-1 had no activity against either acidic (Asp or Glu) or basic(Arg, His, or Lys) amino acid substrates.

EXAMPLE 8 ARTS-1 Endopeptidase Activity Assay

The endopeptidase activity of recombinant GST-ARTS-1 fusion protein wasalso assessed. In this assay, 5 μg of recombinant GST-ARTS-1 fusionprotein was incubated with 10 μg of either bovine serum albumin, humanalbumin, rabbit myosin heavy chain or transferrin overnight. Sample werethen subjected to SDS-PAGE utilizing 4 to 12% gradient gels (Novex) andstained with Coomassie brilliant blue. In this assay, recombinantGST-ARTS-1 was shown to have no demonstrable endopeptidase activityagainst bovine serum albumin, human albumin, rabbit myosin heavy chainor human transferrin.

EXAMPLE 9 Construction of Stably Transfected Cell Lines Expressing Senseand Antisense ARTS-1 cDNA's

In light of the ability of the ARTS-1 polypeptide to bind to the TNFR1ectodomain in the yeast two-hybrid interaction assay, the followingexperiment was conducted in order to determine whether ARTS-1 has theability promote the cleavage and shedding of the TNFR1 ectodomain fromthe surface of human cells in culture. This experiment was done in twophases. The first phase involved construction of stably transfected celllines which expressed either reduced or elevated levels of ARTS-1polypeptide. The NCI-H292 cell line was stably transfected with one ofthree constructs, all based on the pTarget vector (Promega).Transfection was by the Fugene system (Roche). The pTarget vectorexpresses a gene product (encoded by the neo gene) which impartsresistance to the antibiotic G-418, which kills both prokaryotic andeukaryotic cells. The construct also contains a constitutively activeCMV promoter which will express cloned DNA inserts in mammalian cells.The pTarget vectors used to transfect the NCI-H292 cells were:

1) an empty pTarget vector,

2) pTarget vector containing the full length ARTS-1 cDNA coding regionin the sense orientation,

3) pTarget vector containing ARTS-1 cDNA bases 61 to 213 in theanti-sense orientation. This region overlaps the putative transcriptionstart site and intracellular and transmembrane domains. Following thetransfection of these constructs into the host cells, the transfectantswere cultured under selective pressure in RPMI-1640 media supplementedwith 10% heat-inactivated fetal calf serum and 1× antibiotic-antimycotic(Biofluids) and 500 μg/ml of the G-418 (Promega). Two independent clonesof stable transfectants containing either sense or anti-sense ARTS-1plasmid were then generated via limiting dilutions. Both sense andanti-sense clones were screened by immunoblotting (described above)utilizing anti-ARTS-1 polyclonal serum to select duplicate clones forsubsequent analysis.

Cell lines were then selected based upon enhanced or suppressed ARTS-1protein expression as determined by the immunoblotting of cell membranefractions, as shown in FIG. 7. Integration of the empty pTarget vector(indicated “Mock”) had little effect on endogenous ARTS-1 expressioncompared to cells that do not contain any stably integrated plasmid(WT). The two cell lines expressing the full length ARTS-1 cDNA in thesense orientation (ARTS-1) showed a significant increase in ARTS-1protein expression, while the two cell lines expressing ARTS-1 antisensesequence (AS) showed significant reduction in ARTS-1 protein expression.These stably integrated cell lines were examined for sTNFR1 sheddingactivity (See, Example 11, below).

EXAMPLE 10 Construction of Stably Transfected Cell Lines ExpressingMutant ARTS-1 cDNA's

The predicted ARTS-1 polypeptide contains a peptidase/protease consensusmotif found in the aminopeptidase family of gluzincin zincmetalloproteases. It was determined if this peptidase/protease catalyticmotif was necessary for the ability of ARTS-1 to promote shedding of theTNFR1 ectodomain. To conduct this experiment, a series of ARTS-1 mutantswere constructed which contain point mutations predicted to abolish theARTS-1 peptidase/protease activity (Devault et al., FEBS Lett.,23154–23158 [1988]; Devault et al., J. Biol. Chem., 263:4033–4040[1988]; Vallee and Auld, FEBS Lett., 257:138–140 [1989]; Vallee andAuld, Biochemistry 29:5647–5659 [1990]; Wang and Cooper, Proc. Natl.Acad. Sci. USA 90:1222–1226 [1993]). These mutations lie within the zincmetalloprotease family zinc-binding/catalytic domain consensusHEXXH(Y)₁₈E (SEQ ID NO:10; consisting of a zinc binding and catalyticsite domains). In the ARTS-1 polypeptide, this motif is located atH³⁵³ELAH(Y)₁₈E³⁷⁶ (SEQ ID NO:11). The mutations made were H353P, E354V,H353P and E354V in combination, and H357V. Mutagenesis of the ARTS-1gene open reading frame was performed using a QuikChange Site-DirectedMutagenesis Kit (Stratagene) according to the manufacturer'sinstructions.

The NCI-H292 cell line was stably transfected with one of sixconstructs, all based on the pTarget expression vector. The method usedto produce stably transfected cell lines containing these constructs isthe same as that provided in Example 9. These constructs (and resultingcell lines) were:

1) an empty pTarget vector,

2) the ARTS-1 cDNA (WT) coding region,

3) the ARTS-1 cDNA encoding a H353P mutation,

4) the ARTS-1 cDNA encoding a E354V mutation,

5) the ARTS-1 cDNA encoding a H353P and E354V double mutation, and

6) the ARTS-1 cDNA encoding a H357V mutation.

EXAMPLE 11 TNFR1 Ectodomain Shedding Assay

The amount of TNFR1 ectodomain shedding occurring in each of the stablytransfected cell lines described in Example 9 was assessed. The levelsof sTNFR1 ectodomain in cell culture supernatants from these cell lineswere assayed by a commercially available sandwich-enzyme-linkedimmunosorbent assay (ELISA) technique (R & D Systems) with a lower limitof detection of 7.8 pg/ml. The protocol used was according to themanufacturer's instructions. The results of this assay are depicted inFIG. 8 as the mean of 5 independent experiments, with accompanying SEM(standard error of the mean) indicated above the bar. As can be seen inFIG. 8, the cell lines with increased ARTS-1 protein expression alsoshowed a significant increase in the amount of sTNFR1 present in cellculture supernatants as compared to cells transfected with the emptypTarget vector (Mock) or nontransfected (WT) controls. This change insTNFR1 concentration represents an approximately 200% increase in sTNFR1shedding resulting from overexpression of the ARTS-1 protein.

Conversely, cell lines showing decreased ARTS-1 protein expressionshowed significantly decreased levels of sTNFR1 in cell culturesupernatants as compared to cells transfected with the empty pTargetvector, as shown in FIG. 8. This change in concentration represents anapproximately 80% decrease in sTNFR1 shedding resulting from expressionof an ARTS-1 anti-sense transcript encompassing the putative translationstart site.

A similar experiment analyzing the ability of ARTS-1 overexpression topotentiate the cleavage and shedding of TNFR ectodomain from the surfaceof NCI-H292 cells in response to PMA stimulation using these same celllines is shown in FIG. 9. Cell lines overexpressing full length ARTS-1mRNA were stimulated with 0.1 μM phorbol 12-myristate 13-acetate (PMA),which has previously been shown to upregulate sTNFR1 shedding inNCI-H292 cells (Levine et al., Am. J. Respir. Cell Mol. Biol.,14:254–261 [1996]). As shown in FIG. 9, the cell line containing onlythe empty pTarget vector showed only a modest increase in sTNFR1shedding following 24 hours of PMA treatment, increasing fromapproximately 300 pg/ml to 415.3±4.5 pg/ml. However, the cell lineoverexpressing the ARTS-1 cDNA showed a more dramatic increase in sTNFR1shedding following 24 hours of PMA treatment, increasing from 485±16.9pg/ml to 914.2±9.5 pg/ml

This assay was further used to measure the sTNFR1 ectodomain sheddingactivity of ARTS-1 peptidase/protease catalytic mutants. Theconstruction of these mutations (and resulting mutant cell lines) aredescribed in Example 10. The ability of theses mutants to regulatesTNFR1 shedding was ascertained by measuring the concentration of sTNFR1in the cell culture supernatant as described above in this Example.These results are depicted in FIG. 10 as the mean of five independentexperiments, with accompanying SEM (standard error of the mean).

As shown in FIG. 10, the cell line overexpressing the ARTS-1 cDNA(ARTS-1) showed a significantly elevated level of sTNFR1 in the culturesupernatant compared to control cell lines containing no integrated DNA(WT) or containing the empty pTarget vector (MOCK). Unexpectedly, eachof the cell lines containing mutant forms of the ARTS-1 polypeptideshowed elevated levels of sTNFR1 compared to the control lines (i.e., WTand MOCK). This experiment demonstrates the unexpected property wherethe peptidase/protease activity of the ARTS-1 polypeptide is notrequired for the sTNFR1 shedding regulatory activity of ARTS-1polypeptide.

Although not described here, IL-1 and IL-6 receptor ectodomain sheddingcan also be measured by ELISA-based assays using commercially availablekits that measure soluble forms of the IL-1 and IL-6 receptors (R & DSystems, Catalog numbers DR1B00 and DR600, respectively). TheseELISA-based assays measure the concentration of sIL-1RII and sIL-6R,both with a lower limit of detection of 31 pg/ml.

EXAMPLE 12 Effect of ARTS-1 Expression on Membrane-Associated TNFR1

The degree of TNFR1 ectodomain shedding as a function of ARTS-1 proteinexpression was also indirectly assessed by determining the relativeamounts of membrane-bound TNFR1 fragment in each of the stablytransfected cell lines described in Example 9 using Westernimmunoblotting.

Crude membrane fractions from the stably transfected NCI-H292 cellsdescribed in Example 9 were prepared and protein concentrations werequantitated, as described above. Samples from these membrane-derivedprotein preparations were resolved by SDS-PAGE and analyzed by Westernimmunoblotting as described above in Example 5 using a murine anti-humanTNFR1 monoclonal primary antibody which detected the membrane fragmentof the receptor (R & D System). This Western blot is shown in FIG. 11.Two independent strains of each cell line were analyzed in parallel.

As shown in FIG. 11, cell lines over-expressing ARTS-1 (ARTS-1)demonstrated a decrease in membrane-associated TNFR1 relative tonon-transfected (WT) or control transfected (Mock) cell lines,consistent with an increase in constitutive TNFR1 ectodomain shedding.Conversely, cell lines expressing anti-sense ARTS-1 mRNA (AS)demonstrated an increase in membrane-associated TNFR1 relative tonon-transfected (WT) or control transfected (Mock) cell lines,consistent with a reduction in constitutive TNFR1 ectodomain shedding.

EXAMPLE 13 ARTS-1/TNFR1 in vivo Co-Immunoprecipitation Assays

The ability of ARTS-1 to directly interact with TNFR1 ectodomain wasassessed in vivo using a co-immunoprecipitation assay. This assayutilized anti-ARTS-1 antiserum and monoclonal anti-TNFR1 antibodies. Theimmunoprecipitated proteins were visualized by Western immunoblotting.

Crude membrane fractions from cultured NCI-H292 cells were prepared andprotein concentrations were quantitated as described above. From thesemembrane-derived protein preparations, 200 μg samples were incubatedwith 20 μg of murine anti-human TNFR1 monoclonal antibody (R & D System)or 1 ml of anti-ARTS-1 antiserum overnight at 4° C. inimmunoprecipitation buffer (50 mM Tris-HCl, 120 mM NaCl, 0.1% TritonX-100 and COMPLETE™ protease inhibitor (Roche), pH 7.2). Following theincubation, the resulting antibody complexes were immunoprecipitated bybinding to 200 μl of immobilized protein A/G beads (Pierce) for 2 hoursat room temperature. Proteins contained in the samples were thenresolved by SDS-PAGE and analyzed by Western immunoblotting as describedabove.

Two different combinations of precipitation and immunoblotting antibodywere used. The results of these immunoprecipitation experiments areshown in FIG. 12. In one experiment (FIG. 12, top panel), the anti-TNFR1antibody was used in the immunoprecipitation (indicated as “IP” in theFigure), and the anti-ARTS-1 antiserum was used as the primary antibodyin the immunoblotting (indicated as “IB” in the Figure). In a secondexperiment (FIG. 12, bottom panel), the antibodies were reversed, wherethe anti-ARTS-1 antiserum was used in the immunoprecipitation, while theanti-TNFR1 antibody was used as the primary antibody in theimmunoblotting. Also in these experiments, anti-ARTS-1 pre-immune serum(written “PI”) and a purified murine IgG1 isotype (written “IgG1”) wereused as negative controls.

As shown in the top panel of FIG. 12, immunoprecipitation of theNCI-H292 cell membrane proteins with an anti-TNFR1 monoclonal antibodyresulted in the coprecipitation of the 100 kDa ARTS-1 species and,conversely, as seen in the bottom panel, immunoprecipitation withanti-ARTS-1 antiserum coprecipitated the 55 kDa TNFR1. These resultsindicate an in vivo protein-protein interaction between ARTS-1 and TNFR1proteins.

Similar immunoprecipitation experiments were also performed using thestably-transfected NCI-H292 cell lines described in Example 9. In theseexperiments, the anti-TNFR1 antibody was used to immunoprecipitateprotein from the various membrane protein fractions, and the resultingimmunoprecipitate was examined by Western immunoblotting usinganti-ARTS-1 antiserum as the primary antibody. As shown in FIG. 13,immunoprecipitation using an anti-TNFR1 monoclonal antibody of cellmembrane protein derived from the anti-sense ARTS-1 cell line (AS)showed decreased amounts of ARTS-1 protein as compared tocontrol-transfected (Mock) or non-transfected (WT) cells, consistentwith decreased ARTS-1 protein expression in anti-sense ARTS-1 cells. Noincrease in ARTS-1 protein levels relative to control cell lines wasdetected following immunoprecipitation of ARTS-1 overexpressing celllines with an anti-TNFR1 monoclonal antibody, which likely reflectsincreased TNFR1 shedding related to ARTS-1 over-expression.

EXAMPLE 14 Methods for Drug Screening

The present invention provides two examples of methods for drugscreening. These methods identify test compounds which are able toregulate ARTS-1 protein expression in a tissue, or regulate sTNFR1shedding in a cell culture system.

In the first method, cultured human NCI-H292 pulmonary mucoepidermoidcarcinoma cells were exposed to a test compound, in this case,4b-phorbol 12-myristate 13-acetate (PMA) at a concentration of 0.1 μM.Cells were harvested and the membrane protein fraction isolated atintervals from prior to exposure to the test compound (Time=0) to 24hours following exposure to the compound. The protein samples wereanalyzed in a Western immunoblot using the anti-ARTS-1 antiserum at theprimary detection antibody, as described in Examples 4 and 5. Theprotein fractions were analyzed in duplicate, and the relativedensitometry units for each lane are shown beneath the columns. Eachimmunoblot is representative of 3 independent experiments. The resultsof this screening are shown in FIG. 14, Panel A. As can be seen in theFigure, treatment of the cultured cells resulted in an increase inARTS-1 protein expression over the course of 24 hours. Thus, thisscreening method identified a compound that is a candidate for furtherdrug development.

Results from a second screening method are illustrated in FIG. 14, PanelB. In this screening method, cultured human NCI-H292 pulmonarymucoepidermoid carcinoma cells were again exposed to a test compound,PMA, at a concentration of 0.1 μM. Culture medium supernatant wascollected from the cultures at intervals between 0 and 24 hours posttreatment. The samples were analyzed by the TNFR1 ectodomain sheddingassay using an ELISA as described in Example 11. In this assay,anti-sTNFR1 antibody was used to quantitate the concentrations of sTNFR1in the cell culture supernatants. The results of this ELISA screeningare shown in FIG. 14, Panel B. As can be seen in this Figure, treatmentof the cultured cells resulted in an increase in sTNFR1 shedding overthe course of 24 hours compared to a control culture (n=5,*P) <0.05).Thus, this screening method also identified a compound that is acandidate for further drug development.

EXAMPLE 15 Tumor Necrosis Factor Bioactivity (Cytotoxicity) Assay

The bioactivity of tumor necrosis factor (TNF) is measured by a cellcytotoxicity assay utilizing the WEHI 164 clone-13 mouse fibrosarcomacell line (ATCC, CRL 1751). This cell line has been shown to be highlysensitive to the cytotoxic effects of human tumor necrosis factor atconcentrations as low as 0.1 pg/ml TNF following pretreatment withactinomycin D (Eskandari et al., Immunol. Invest., 19:69–79 [1990]). Inthis assay, WEHI 164 cells are seeded into 96-well microtiter plates ata density of 40,000 cells per well in RPMI-1640 supplemented withpenicillin (100 units/ml) (Advanced Biotechnologies), streptomycin (100μg/ml) (Advanced Biotechnologies), L-glutamine (2 mM) (AdvancedBiotechnologies), 10% heat-inactivated fetal-calf serum (Inovar), andactinomycin-D (0.5 μg/ml) (Calbiochem). Test samples (serum, plasma orany other body fluid) to be tested for TNF activity are diluted 6-foldin RPMI-1640 culture medium (Advanced Biotechnologies), heat-inactivatedat 56° C. for 30 minutes, and sterile filtered. A volume of 50 μl of thediluted, heat-inactivated sample is added to the microtiter plate wellscontaining the WEHI cells. Duplicate test samples are incubated in thepresence of polyclonal rabbit anti-human TNF antiserum (Genzyme) orcontrol rabbit serum (Life Technologies). Cells are incubated for 20hours, after which time 20 μl of a 5 mg/ml stock solution of thetetrazolium salt 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbromide (also called MTT or Thiazolyl blue; Sigma, Catalog Number M2128)in phosphate buffered saline (PBS) is added to each well. Following anadditional 4 hours incubation, the plates are spun at 950×g for 10minutes, the media from each well is aspirated, and 100 μl of 0.04NHCl/2-propanol is added to each well, and the plates are incubatedovernight in the dark at room temperature. In the morning, an additional100 μl of 0.04N HCl/2-propanol is added to each well and incubated fortwo hours in the dark at room temperature. Cell survival is thenmeasured colorimetrically using a microplate reader with a 570 nmwavelength test filter and a 630 nm reference filter. Cell survival ineach test well is determined as a percentage of the optical density ofcontrol wells. TNF-specific killing is defined as the difference in cellkilling with or without the anti-TNF antiserim and is compared withstandard curves produced with recombinant human TNF-α (R & D Systems).

EXAMPLE 15 Statistical Analysis

Data are presented as mean±standard error of the mean. Comparisons weremade utilizing a paired two-tailed student's T test with a Bonneferonicorrection for multiple comparisons. A P value<0.005 was consideredsignificant.

These experiments demonstrate that expression of ARTS-1 protein directlycorrelates with and is necessary for soluble TNFR1 ectodomain shedding.Furthermore, ARTS-1 represents the first gene and protein which havebeen identified which have the ability regulate TNTFR1 ectodomainshedding. In addition, the present invention provides the nucleic acidand amino acid sequences of this gene and protein.

Furthermore, the compositions and methods of the present inventionprovide therapeutic applications for the treatment of a wide variety ofdisorders of the immune system which arise as a result of improper TNFactivity.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described methods and compositions of the present invention willbe apparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology and immunology and/or related fieldsare intended to be within the scope of the present invention.

1. A method for promoting the shedding of the extracellular domain of atleast one cytokine receptor, comprising the steps of: a) providing: i) apolypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, and ii) one or more cells expressing a cytokine receptor on theirplasma membrane extracellular surface, wherein the cytokine receptor isselected from the group consisting of a type-1 tumor necrosis factorreceptor, a type I interleukin-1 cytokine receptor, a type IIinterleukin-1 cytokine receptor, and an interleukin-6 cytokine receptoralpha-chain gp80; and b) delivering said polypeptide to said one or morecells, whereby said polypeptide promotes the shedding of the cytokinereceptor from the surface of said cells.
 2. The method of claim 1,wherein said cytokine receptor is a type-1 tumor necrosis factorreceptor.
 3. The method of claim 1, wherein said cytokine receptor is atype I interleukin-1 cytokine receptor.
 4. The method of claim 1,wherein said cytokine receptor is a type II interleukin-1 cytokinereceptor.
 5. The method of claim 1, wherein said cytokine receptor is aninterleukin-6 cytokine receptor alpha-chain gp80.
 6. The method of claim1, wherein the one or more cells are cultured cells.
 7. The method ofclaim 1, wherein the one or more cells are human cells.
 8. The method ofclaim 2, wherein the one or more cells are human cells.
 9. The method ofclaim 3, wherein the one or more cells are human cells.
 10. The methodof claim 4, wherein the one or more cells are human cells.
 11. Themethod of claim 5, wherein the one or more cells are human cells. 12.The method of claim 1, wherein the one or more cells are in a tissue.13. The method of claim 2, wherein the one or more cells are in atissue.
 14. The method of claim 3, wherein the one or more cells are ina tissue.
 15. The method of claim 4, wherein the one or more cells arein a tissue.
 16. The method of claim 5, wherein the one or more cellsare in a tissue.
 17. The method of claim 7, wherein the one or morecells are in a tissue.